Cyclooxygenase (COX; prostaglandin endoperoxide synthase, EC 1.14.99.1) catalyzes the conversion of arachidonic acid to prostaglandin (PG) endoperoxide (PGH). This is the rate-limiting step in PG and thromboxane biosynthesis. Two isoforms of COX have been cloned from various animal cells: constitutively expressed COX-1 (1, 2, 3, 4, 5) and mitogen-inducible COX-2 (6, 7, 8, 9, 10, 11) . Prostaglandins produced as a result of the activation of COX-1 may have some physiological functions, such as the antithrombogenic action of prostacyclin released by the vascular endothelium, and cytoprotective effect of PGs produced by the gastric mucosa(12) . COX-2 is expressed following the activation of cells by various proinflammatory agents including cytokines(9, 11, 13) , endotoxin(14) , and other mitogens(7, 8, 9) . These observations lead to the suggestion that COX-2 instead of COX-1 may be responsible for producing prostanoids involved in inflammation and/or mitogenesis. Furthermore, the ability of nonsteroidal anti-inflammatory drugs (NSAIDS) to inhibit COX-2 may well explain their therapeutic efficacy as anti-inflammatory drugs, whereas inhibition of COX-1 may explain their unwanted side effects(15) .

For this reason, considerable interest has been focused on finding NSAIDS which can selectively inhibit COX-2 as more desirable anti-inflammatory agents with minimum side effects. While these NSAIDS inhibit the enzyme activity of endogenous COX, glucocorticoids are known to suppress COX-2 expression at transcriptional and/or translational steps without significant effect on COX-1 expression (14, 15, 16, 17) . Other than glucocorticoids, pharmacological agents that can suppress the expression of COX-2 without affecting that of COX-1 have not been reported. Neoplastic transformation of chicken embryo fibroblasts by Rous sarcoma virus results in the activation of a set of early response genes encoding growth factors and transcription factors involved in the regulation of cell division(18, 19) . One of these genes encodes the mitogen-inducible cyclooxygenase (COX-2). Induction of these genes is dependent on the activity of the v-Src oncogene product p60, a tyrosine kinase. Radicicol, a fungal antibiotic with a macrocyclic ring structure(20, 21) , was reported as an inhibitor of p60 tyrosine kinase(22) . It has been demonstrated that lipopolysaccharide (LPS) rapidly increases protein tyrosine phosphorylation in macrophages, and that this early signaling event appears to mediate some downstream macrophage responses to LPS(23, 24, 25) . For this reason, we investigated whether radicicol inhibits the expression of COX-2 in rat alveolar macrophages stimulated with LPS. We report here that radicicol suppresses protein tyrosine phosphorylation and the expression of COX-2 (IC, 27 nM) without affecting COX-1 expression in LPS-stimulated alveolar macrophages in rats.

MATERIALS AND METHODS

Preparation of Radicicol

Figure 1: Chemical structure of radicicol.

Isolation of Macrophages

Preparation of Cell Lysates and Antiphosphotyrosine Immunoblotting

To locate Src family kinases and mitogen-activated protein kinases, the same membrane used for antiphosphotyrosine immunoblot was stripped in the buffer (2% SDS, 50 mM Tris-HCl, 100 mM 2-mercaptoethanol, pH 6.5 at 50 °C for 30 min). The stripped membrane was reprobed with polyclonal anti-mitogen-activated protein kinase antibodies (UBI, 1 µg/2 ml in PBS containing 0.1% Tween 20 and 3% nonfat dry milk) or polyclonal anti-Src family kinase antibodies (Santa Cruz, 1 µg/ml), followed by goat anti-rabbit IgG coupled to horseradish peroxidase (1 µg/10 ml). The immunoreactive protein bands were visualized by the ECL detection system.

Immunoprecipitation

In Vitro p60 Kinase Assay

Assay for Cyclooxygenase

Effects of radicicol on enzyme activities of COX-1 and COX-2 purified from ram seminal vesicle and sheep placenta, respectively, (Cayman Chemicals) were determined by the conversion of [C]arachidonic acid (Dupont, specific activity, 57 mCi/mmol) to PGE after separation by thin layer chromatography (TLC) as described by Mitchell et al.(30) . The reaction mixture (1 ml) in 50 mM Tris buffer (pH 8.0) contained arachidonic acid (10.88 µM) together with [C]arachidonic acid, glutathione (5 mM), epinephrine (5 mM), and hematin (1 µM). The reactions were initiated by adding the purified enzymes and incubated at 37 °C for 10 min. PGs were separated in TLC in a solvent of the organic phase of ethyl acetate/trimethylpentane/acetic acid/water, 110:50:20:100 (v/v). The PGE band was identified by a cold standard run side by side and visualized in an iodine tank. Radioactive PGE bands were identified by superimposing the autoradiograph run on TLC plates and then scraped off. The radioactivity of the PGE band was determined by scintillation counting.

Metabolic Labeling and Immunoprecipitation

Western Blot Analysis for COX-1 Protein

RNase Protection Assay

Measurement of COX mRNA and PG Levels in Glomeruli and Whole Kidney in Rats with Experimental Glomerulonephritis (GN)

RESULTS AND DISCUSSION

Inhibition of Protein Tyrosine Phosphorylation by Radicicol in LPS-stimulated Macrophages

Figure 2: Time course of protein tyrosine phosphorylation and its inhibition by radicicol in LPS-stimulated macrophages. A, cells were pretreated with radicicol (200 ng/ml) for 4 h and then stimulated with LPS (10 µg/ml) containing radicicol or vehicle, dimethyl sulfoxide (DMSO, 5 µl/ml) for the various time periods indicated. Solubilized proteins were analyzed by antiphosphotyrosine immunoblotting as described under ``Materials and Methods.'' Molecular size markers run on the center lane between dimethyl sulfoxide, and radicicol lanes are shown on the left. An arrow on the right indicates tyrosine-phosphorylated protein bands superimposed with p53/56 bands shown below. B, the same membrane used for antiphosphotyrosine immunoblot in A was stripped and reprobed with polyclonal antimitogen-activated protein kinase antibodies (UBI) recognizing both MAPK-1 and MAPK-2. The time scale is the same as that for A. C, the same sample used for A was immunoblotted with antiphosphotyrosine antibody, stripped, and reprobed with polyclonal anti-p53/56 antibodies. The time scale is the same as that for A.

Figure 3: Time course for the expression of COX activity induced by LPS and its inhibition by radicicol in macrophages pretreated with aspirin. Rat alveolar macrophages were allowed to attach for 2.5 h in the presence of aspirin (500 µM) to inactivate endogenous cyclooxygenase, washed three times, and then incubated in RPMI with LPS (10 µg/ml) in the presence or absence of radicicol (200 ng/ml). After removing the media, cells were incubated with arachidonic acid (30 µM) for 10 min. The levels of PGE produced from exogenous arachidonic acid were measured by radioimmunoassay to determine the activity of de novo synthesized cyclooxygenase. The value for 2.5 h indicates the endogenous COX activity prior to aspirin treatment. Values for each time point are the mean of three samples.

Figure 5: Time course for levels of mRNA for COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase (GAP) in LPS-treated macrophages in the presence or absence of radicicol. Rat alveolar macrophages were incubated with LPS in the presence or absence of radicicol (100 ng/ml) for specified times. Total RNA was extracted and hybridized with riboprobes, as described under ``Materials and Methods.'' The [-P]UTP-labeled COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase were protected with the complementary mRNA from macrophages. The gel was scanned (AMBIS), and the radioactivities of COX-1 and COX-2 bands were factored relative to that of the glyceraldehyde-3-phosphate dehydrogenase band.

Radicicol Inhibits Tyrosine Phosphorylation of Src Family Tyrosine Kinases

Figure 6: Western blot analyses of pooled cell lysates used in Fig. 2for individual Src family tyrosine kinase. Membranes used for antiphosphotyrosine immunoblot were stripped and reprobed with polyclonal anti-p60, p58/64, p53/56, or p59 antibodies. P, antiphosphotyrosine immunoblot of pooled lysates; S, stripped membrane reprobed with anti-p60 antibodies; H, stripped membrane reprobed with anti-p58/64 antibodies; L, stripped membrane reprobed with anti-p53/56 antibodies; F, stripped membrane reprobed with anti-p59 antibodies. Molecular size markers are in kilodaltons.

Antiphosphotyrosine immunoblot of immunoprecipitates of cell lysates indicated that p53/56 and p59 were the major tyrosine phosphorylated Src family tyrosine kinases (Fig. 7). When the cell lysates after the immunoprecipitation were analyzed by antiphosphotyrosine immunoblot, the lower band superimposing with p53/56 disappeared completely in the sample for which p53/56 was removed by immunoprecipitation with anti-p53/56 antibodies (Fig. 8). It has been demonstrated that LPS induces rapid tyrosine phosphorylation of isoforms of mitogen-activated protein kinases in elicited murine peritoneal exudate macrophages and RAW 264.7 cells(24) . Both MAPK-1 (42 kDa) and MAPK-2 (44 kDa) were detected in cell lysates derived from the time course study as determined by Western blot analysis using polyclonal anti-rat MAP kinase antibodies recognizing both MAPK-1 and MAPK-2 (UBI, Fig. 2B). However, tyrosine-phosphorylated mitogen-activated protein kinases were not detected by the antiphosphotyrosine immunoblotting procedure used in our studies (Fig. 2A).

Figure 7: Antiphosphotyrosine immunoblot analyses of individual Src tyrosine kinase immunoprecipitated from pooled cell lysates used in Fig. 2. P, pool cell lysate; S, immunoprecipitate with anti-p60 antibodies; H, immunoprecipitate with p58/64 antibodies; L, immunoprecipitate with p53/56 antibodies; F, immunoprecipitate with p59 antibodies. Molecular size markers are in kilodaltons.

Figure 8: Antiphosphotyrosine immunoblot analyses of cell lysates after immunoprecipitating individual Src family tyrosine kinase as described in the legend for Fig. 7. P, pooled cell lysate without immunoprecipitation; S, cell lysate after depleting p60 by immunoprecipitation; H, cell lysate after depleting p58/64 by immunoprecipitation; L, cell lysate after depleting p53/56 by immunoprecipitation; F, cell lysate after depleting p59 by immunoprecipitation. Molecular size markers are in kilodaltons.

Pretreatment of macrophages with radicicol in our studies resulted in suppression of levels of tyrosine-phosphorylated proteins in time-dependent and dose-dependent fashions (Fig. 2A and 9A). Pretreatment of macrophages with radicicol or herbimycin A resulted in a significant reduction of levels of p53/56 (Fig. 9B). This reduction in levels of p53/56 was correlated to suppressed levels of tyrosine-phosphorylated proteins by radicicol or herbimycin A both in the time course (Fig. 2A) and the dose-response studies (Fig. 9A). Pretreatment of macrophages with radicicol not only suppressed the basal levels (zero time) of tyrosine-phosphorylated proteins, but also attenuated the stimulatory effect of LPS on protein tyrosine phosphorylation (Fig. 2A). Some Src family tyrosine kinases, in association with cell surface proteins, participate in normal signaling pathways in hemopoietic cells including monocytes and macrophages(35) . It has been demonstrated that initial interaction of LPS with monocytes and macrophages involves a LPS-binding protein that binds to LPS and a glycosylphosphatidylinositol-anchored cell-surface glycoprotein, CD14(25) . It was also shown that p53/56 was co-immunoprecipitated with CD14 in human monocytes(23) . This suggests a critical role of Src family tyrosine kinases in the LPS/CD14-mediated signal transduction pathway in monocytes and macrophages. Our results shown in Fig. 6Fig. 7Fig. 8indicate that Src family tyrosine kinases are the major tyrosine-phosphorylated proteins, and that p53/56 is one of the major tyrosine-phosphorylated Src family tyrosine kinases in rat alveolar macrophages. These results are consistent with the results showing that p53/56 is the major Src family tyrosine kinase which is associated with CD14 in human monocytes(23) . Furthermore, radicicol inhibited tyrosine phosphorylation and levels of p53/56 in a time-dependent and dose-dependent fashion (Fig. 2, A and B, and 9, A and B). Inhibitory effects of radicicol on protein tyrosine phosphorylation required pretreatment of cells with radicicol for 4 h prior to LPS stimulation. Such pretreatment of cells was also required for herbimycin A in our studies and in studies by other investigators(24, 25) .

Figure 9: Dose-response by radicicol in inhibiting protein tyrosine kinase in LPS-stimulated macrophages. Inhibitory effects of radicicol and herbimycin A on tyrosine-specific protein phosphorylation in LPS-stimulated macrophages were determined by antiphosphotyrosine immunoblotting as described under ``Materials and Methods.'' A, cells were pretreated with indicated concentrations of radicicol or herbimycin A for 4 h and then stimulated with LPS (10 µg/ml) containing radicicol or herbimycin A. The control was pretreated with vehicle, dimethyl sulfoxide (5 µl/ml) only. An arrow on the right indicates tyrosine-phosphorylated protein bands superimposed with p53/56 bands shown below. Rad, radicicol; Herb, herbimycin A. B, Western blot analysis of p53/56 in the same samples used in A as described in the legend for Fig. 2C. LYN, p53/56. Molecular size markers are shown in kilodaltons.

In vitro kinase assay using purified p60 kinase (UBI) and p60 kinase substrate peptide revealed that radicicol inhibits the kinase activity at much higher concentrations (IC = 8.2 µM, Fig. 10) than those required to suppress protein tyrosine phosphorylation in LPS-stimulated macrophages (Fig. 9A) or those required to suppress the expression of COX-2 (Fig. 11). Similarly, concentrations of herbimycin A needed to suppress COX-2 expression and protein tyrosine phosphorylation in LPS-stimulated macrophages were much lower than those known to be required to inhibit p60 kinase activity in vitro. The IC value of herbimycin A for the in vitro kinase activity was reported as 12 µM(36) .

Figure 10: Inhibition of p60 tyrosine kinase activity by radicicol. In vitro kinase assay was carried out using purified p60, p60 kinase substrate peptide, and [-P]ATP as described under ``Materials and Methods.'' Values are mean ± S.E. of three samples.

Figure 11: The dose-response by radicicol in inhibiting the recovery of COX activity and 5-lipoxygenase (5-LO) activity in LPS-stimulated macrophages. Rat alveolar macrophages pretreated with aspirin as described in the legend for Fig. 3were incubated in RPMI with LPS and various concentrations of radicicol for 16 h. The activity of de novo synthesized COX was determined by measuring the levels of PGE produced from exogenous arachidonic acid as described in the legend for Fig. 3. The activity of 5-lipoxygenase was determined by measuring the levels of 5-hydroxyeicosatetraenoic acid by radioimmunoassay. Values for each dose are the means of three to six samples.

Together, these results suggest that radicicol suppresses tyrosine phosphorylation of Src family tyrosine kinases in LPS-stimulated macrophages both by decreasing levels of enzyme proteins and inhibiting the enzyme activity. It has been shown that herbimycin A increased the degradation of p60(37) . However, the possibility that radicicol and herbimycin A inhibit expression of the tyrosine kinases during the pretreatment period cannot be ruled out.

The mechanism by which radicicol inhibits protein tyrosine kinases is not known. It has been speculated that herbimycin A inactivates p60 kinase by irreversibly binding to SH group(s) of p60. This speculation was based on the fact that herbimycin A is readily inactivated by sulfhydryl compounds. These compounds abolished the inhibitory effect of herbimycin A on p60 kinase activity. It was postulated that this inactivation occurs through conjugation between highly polarized double bonds in the benzoquinone moiety of herbimycin A and the SH group of sulfhydryl compounds or Src kinases. Similarly, conjugation of SH group can occur at C-9 with a conjugated double bond or at C-5 and C-6 bearing an epoxide in the radicicol molecule as shown in Fig. 1. It was observed that dithiothreitol abolished the ability of radicicol to block p60 kinase activity(22) . However, there is no evidence as to whether sulfhydryl compounds also abolish effects of radicicol propagated to steps downstream of Src tyrosine kinases.

Inhibition of the Recovery of Cyclooxygenase Enzyme Activity by Radicicol in Macrophages Pretreated with Aspirin

The recovery of COX activity in aspirin-pretreated and LPS-stimulated macrophages started only after 6 h of incubation (Fig. 3). This time course paralleled the time course of de novo synthesized COX-2 protein as shown in our previous studies(14) . COX activity recovered after 16 h of incubation was always greater than COX activity of unstimulated cells prior to aspirin treatment, although the magnitude of the difference varies with batches of cells. This indicates that the activity of COX-2 expressed as a result of LPS stimulation is much greater than that of COX-1 present in unstimulated cells. This suggests that COX-2 but not COX-1 plays a major role in producing prostanoids in response to inflammatory stimuli in macrophages. The inhibition of COX-2 expression by radicicol was not reversed during the 24-h incubation period.

The dose-response to radicicol in inhibiting the recovery of COX activity showed that the IC is 10 ng/ml (27 nM) as shown in Fig. 11. These doses of radicicol did not significantly affect 5-lipoxygenase activity as determined by measuring 5-hydroxyeicosatetraenoic acid (5-HETE) produced from exogenous arachidonic acid (30 µM). Radicicol suppressed the expression of COX-2 protein but not COX-1 and glyceraldehyde-3-phosphate dehydrogenase (GAP) proteins. The rate of COX-2 protein synthesis, as determined by the immunoprecipitation assay using the specific COX-2 antibodies(14) , showed a parallel dose-dependent inhibition by radicicol in macrophages which were metabolically labeled with [S]methionine in the presence of LPS (Fig. 4A). When the same samples were precleared with COX-2 antiserum to remove COX-2 protein and then immunoprecipitated with COX antibodies which preferentially recognize COX-1 protein, no COX bands were detected in the autoradiograph (figure not shown). This result indicates that COX-1 is not synthesized in significant amounts during the 2-h labeling period in LPS-treated macrophages.

Figure 4: Effects of radicicol on expressions of COX-2, COX-1, and glyceraldehyde-3-phosphate dehydrogenase (GAP). A, rat alveolar macrophages were preincubated for 14 h with LPS (10 µg/ml) and various concentrations of radicicol and then further incubated in methionine-free RPMI containing 200 µCi of [S]methionine for 2 h. Cells were lysed in the lysing buffer. Aliquots of the samples with equal amounts of radioactivity were precleared with the preimmune serum, immunoprecipitated with COX-2 antibodies, and subjected to SDS-polyacrylamide gel electrophoresis and fluorography as described under ``Materials and Methods.'' B, rat alveolar macrophages were incubated in RPMI with 3% serum for 16 h with or without LPS and/or radicicol. For Western blot analysis, microsomes from lysed cells were used for COX-1, and whole lysate was used for glyceraldehyde-3-phosphate dehydrogenase. Lane 1, cells incubated without LPS and radicicol; lane 2, cells incubated with LPS (10 µg/ml) only; lane 3, cells incubated with LPS and radicicol (50 ng/ml); lane 4, cells incubated with LPS and radicicol (200 ng/ml).

Levels of COX-1 and glyceraldehyde-3-phosphate dehydrogenase proteins were assessed by Western blot analysis (Fig. 4B). Levels of these enzymes in macrophages were not affected by either LPS or radicicol. Both COX-1 and glyceraldehyde-3-phosphate dehydrogenase are products of housekeeping genes whose expression, normally, is not stimulated by mitogens.

Radicicol also inhibited the LPS-induced expression of COX-2 in rat smooth muscle cells and human peripheral blood monocytes. Furthermore, radicicol inhibited the expression of COX-2 induced by IL-1 in rat smooth muscle cells, although the IC value of radicicol for the inhibition of IL-1-induced COX-2 expression was much greater than that for the inhibition of COX-2 expression induced by LPS (Table 1).

Radicicol at concentrations up to 1000 ng/ml did not significantly affect the expression of COX-2 induced by phorbol 12-myristate 13-acetate (PMA) in rat alveolar macrophages (Table 1). It was also shown that another protein tyrosine kinase inhibitor, herbimycin A, only weakly inhibits PMA-induced release of [H]arachidonic acid metabolites in macrophage-like cell line (RAW 264.7), whereas it dramatically inhibits LPS-induced release of [H]arachidonic acid metabolites(24) . It has been demonstrated that PMA-stimulated c-raf activity in human T cells and some of functional effects of PMA were resistant to the inhibitory effects of herbimycin A(38) . These results suggest that protein tyrosine phosphorylation is proximal to protein kinase C activation in these signal transduction pathways.

It has been well documented that endotoxin LPS induces rapid protein tyrosine phosphorylation in macrophages(23, 24, 25) . In addition to radicicol, other tyrosine kinase inhibitors also suppressed LPS-induced expression of COX-2, although IC values of these inhibitors were much greater than that of radicicol (22 µM, 52 nM, and 357 µM for genistein, herbimycin A, and tyrphostin (AG-494), respectively). Earlier, it was shown that PMA-induced expression of COX-2 was not inhibited by radicicol. Together, these results suggest that tyrosine protein phosphorylation is the proximal step in the LPS-induced signal transduction pathway leading to the induction of COX-2 expression in macrophages, and that the inhibitory effect of radicicol on LPS-induced COX-2 expression is due at least in part to the suppression of activities of tyrosine kinases.

Suppression of Steady State Levels of COX-2 mRNA by Radicicol

Figure 12: Dose-response of COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels to radicicol. Rat alveolar macrophages were incubated with LPS in the presence of various concentrations of radicicol for 2 h. Levels of mRNA were determined by RNase protection assay, as described in the legend for Fig. 11.

Figure 13: The effect of radicicol on the stability of COX-1 and COX-2 mRNA. The effect of radicicol on COX-1 and COX-2 mRNA stability was examined by stimulating macrophages with LPS for 2 h in the presence or absence of radicicol (100 ng/ml). Actinomycin D at a final concentration of 2 µg/ml was added to block further transcription, and the levels of COX-1 and COX-2 mRNA were monitored at specified time intervals over a period of 6 h after the addition of actinomycin D by RNase protection assay. Glyceraldehyde-3-phosphate dehydrogenase mRNA, which is known to have a long half-life, was used as an internal control for RNA loading. The radioactivity was quantitated by AMBIS as shown in Fig. 5. Percents of initial levels (cpm of COX-1 or COX-2 10 cpm of glyceraldehyde-3-phosphate dehydrogenase) were plotted against the incubation time after the addition of actinomycin D. A, COX-1 mRNA stability. B, COX-2 mRNA stability.

Radicicol Did Not Affect the Enzyme Activity of Either COX-1 or COX-2

To determine the direct effect of radicicol on the enzyme activity of COX-2, macrophages were pretreated with aspirin (500 µM) for 4 h to inactivate the endogenous COX, washed three times, and then further incubated with LPS for 16 h in order to maximally stimulate COX-2 expression. After removing the medium, cells were incubated with radicicol (100 ng/ml) or with the vehicle (dimethyl sulfoxide) for 1 h, and then COX activity was determined as described above. COX activity in these cells reflects specifically that of COX-2 because endogenous COX-1 was inactivated by aspirin and LPS induces selective expression of COX-2 in macrophages as shown in our previous study(14) . The results showed that the level of PGE produced from exogenous arachidonic acid in cells treated with radicicol was 150.2 ± 9.3 (pg/µg of protein, n = 3), whereas that in cells treated with the vehicle was 121.1 ± 8.8 (pg/µg of protein, n = 3). The enzyme activities of purified COX-1 and COX-2 were also not affected by radicicol (1000 ng/ml). COX-1 activities were 3.82 ± 0.42 and 5.40 ± 0.78 nmol/µg of protein/10 min (n = 4) for the vehicle and radicicol-treated samples, respectively, whereas COX-2 activities were 5.59 ± 0.46 and 5.18 ± 0.90 nmol/µg of protein/10 min (n = 4) for the vehicle and radicicol-treated samples, respectively. These results indicate that radicicol does not directly affect the enzyme activities of COX-1 and COX-2 but it specifically suppresses the expression of COX-2 in LPS-stimulated alveolar macrophages.

The protein tyrosine kinase inhibitor genistein has been shown to inhibit platelet-activating factor-stimulated PGE production in LPS-primed p388D1 macrophage-like cells(39) . Genistein and another protein tyrosine kinase tyrphostin-25 inhibited PGE production in murine resident peritoneal macrophages stimulated with zymosan, calcium ionophore A23187, and PMA(39) . It was shown that these inhibitors had no inhibitory effect on cyclooxygenase activity in the intact macrophages(40) . Therefore, it was speculated that the inhibition of PGE production by protein tyrosine kinase results from reduced arachidonic acid release from membrane lipids by phospholipase A. Specific effects of these inhibitors on the expression of COX were not determined in these studies. Our results showing that protein tyrosine kinase inhibitors suppress the expression of COX-2 suggest that the inhibition of PG production in LPS-stimulated macrophages by these inhibitors is due at least in part to the suppression of COX-2 expression.

In Vivo Suppression of COX-2 Expression by Radicicol in Rats with Experimental Glomerulonephritis (GN)

Figure 14: In vivo effects of radicicol on expressions of COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase in rats with experimental glomerulonephritis. Glomerular injury was induced in rats with nephritogenic doses of anti-glomerular basement membrane (GBM) antibodies, as described under ``Materials and Methods.'' Radicicol or vehicle was infused through the renal artery for 60 min at the dose of 10 µg/100 g body weight per min. The rats were sacrificed after 4 h and 24 h following anti-GBM antibody injection. The total RNA extracted from whole kidneys and glomeruli was used for RNase protection assay to determine levels of COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase mRNA.

In summary, Src family tyrosine kinases were the major tyrosine-phosphorylated proteins in LPS-stimulated macrophages. Radicicol suppressed tyrosine phosphorylation of these kinases. Radicicol suppressed the expression of COX-2, but not COX-1, in LPS-stimulated macrophages and in glomeruli of rats with experimentally induced glomerulonephritis in which COX-2 expression is known to be enhanced. Thus, radicicol is a potent inhibitor for the expression of COX-2 both in vitro and in vivo. Radicicol did not inhibit PMA-induced expression of COX-2. Other protein tyrosine kinases inhibited the COX-2 expression in LPS-stimulated macrophages. These results suggest that the inhibition of COX-2 expression by radicicol is at least in part mediated through the inhibition of protein tyrosine kinases in LPS-stimulated macrophages. The magnitude of the inhibition of COX-2 protein synthesis by radicicol was much greater than that of the steady state levels of COX-2 mRNA. The rate of COX-2 mRNA degradation was slightly increased by radicicol. However, this does not account for the drastic inhibition of the activity and protein levels of COX-2 by radicicol. Taken together, these results suggest that the inhibition of COX-2 expression by radicicol occurs mainly at post-transcriptional steps.

FOOTNOTES

*

This work was supported in part by National Institutes of Health Grant R01 DK-41868 and United States Department of Agriculture Grants 93-37200-8961 (to D. H.) and R01 DK-20043 (to C. B. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence and reprint requests should be addressed: Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Rd., Baton Rouge, LA 70808. Fax: 504-765-2525.

()

The abbreviations used are: COX, cyclooxygenase; PGE, prostaglandin E; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; NSAIDS, nonsteroidal anti-inflammatory drugs; GN, glomerulonephritis; GBM, glomerular basement membrane; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase.

ACKNOWLEDGEMENTS

REFERENCES

1. DeWitt, D. L., and Smith, W. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1412-1416 [Abstract/Free Full Text]
2. Merlie, J. P., Fagan, D., Mudd, J., and Needleman, P. (1988) J. Biol. Chem. 263, 3550-3553 [Abstract/Free Full Text]
3. Yokoyama, C., Takai, T., and Tanabe, T. (1988) FEBS Lett. 231, 347-351 [CrossRef][Medline] [Order article via Infotrieve]
4. DeWitt, D. L., El-Harith, E. A., Kraemer, S. A., Andrews, M. J., Yao, E. F., Armstrong, R. L., and Smith, W. L. (1990) J. Biol. Chem. 265, 5192-5198 [Abstract/Free Full Text]
5. Yokoyama, C., and Tanabe, T. (1989) Biochem. Biophys. Res. Commun. 165, 888-894 [CrossRef][Medline] [Order article via Infotrieve]
6. Xie, W., Chipman, J. G., Robertson, D. L., Erikson, R. L., and Simmons, D. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2692-2696 [Abstract/Free Full Text]
7. Kujubu, D. A., Fletcher, B. S., Varnum, B. C., Lim, R. W., and Herschman, H. R. (1991) J. Biol. Chem. 266, 12866-12872 [Abstract/Free Full Text]
8. O'Banion, M. K., Sadowski, H. B., Winn, V., and Young, D. A. (1991) J. Biol. Chem. 266, 23261-23267 [Abstract/Free Full Text]
9. Hla, T., and Neilson, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7384-7388 [Abstract/Free Full Text]
10. Jones, D. A., Carlton, D. P., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (1993) J. Biol. Chem. 268, 9049-9054 [Abstract/Free Full Text]
11. Feng, L., Sun, W., Xia, Y., Tang, W. W., Chanmugam, P., Soyoola, E., Wilson, C. B., and Hwang, D. (1993) Arch. Biochem. Biophys. 307, 361-368 [CrossRef][Medline] [Order article via Infotrieve]
12. Whittle, B. J. R., Higgs, G. A., Eakins, K. E., Moncada, S., and Vane, J. R. (1980) Nature 284, 271-273 [CrossRef][Medline] [Order article via Infotrieve]
13. Feng, L., Xia, Y., Garcia, G. E., Hwang, D., and Wilson, C. B. (1995) J. Clin. Invest. , in press
14. Lee, S. H., Soyoola, E., Chanmugam, P., Hart, S., Sun, W., Zhong, H., Liou, S., Simmons, D., and Hwang, D. (1992) J. Biol. Chem. 267, 25934-25938 [Abstract/Free Full Text]
15. Vane, J. (1994) Nature 367, 215-216 [CrossRef][Medline] [Order article via Infotrieve]
16. Kujubu, D. A., and Herschman, H. R. (1992) J. Biol. Chem. 267, 7991-7994 [Abstract/Free Full Text]
17. Xie, W., Merrill, J. R., Bradshaw, W. S., and Simmons, D. L. (1993) Arch. Biochem. Biophys. 300, 247-252 [CrossRef][Medline] [Order article via Infotrieve]
18. Simmons, D. L., Levy, D. B., Yannoni, Y., and Erikson, R. L. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1178-1182 [Abstract/Free Full Text]
19. Bedard, P. A., Alcorta, D., Simmons, D. L., Luk, K. C., and Erikson, R. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6715-6719 [Abstract/Free Full Text]
20. Delmonte, P., and Delmonte-Plaquee, J. (1953) Nature 171, 344-345
21. McCapra, F., Scott, A. I., Delmotte, P., and Delmotte-Plaquee, J. (1964) Tetrahedron Lett. 15, 869-875 [CrossRef]
22. Kwon, H. J., Yoshida, M., Fukui, Y., Horinouchi, S., and Beppu, T. (1992) Cancer Res. 52, 6926-6930 [Abstract/Free Full Text]
23. Stefanova, I., Corcoran, M. L., Horak, E. M., Wahl, L. M., Bolen, J. B., and Horak, I. D. (1993) J. Biol. Chem. 268, 20725-20728 [Abstract/Free Full Text]
24. Weinstein, S. L., Gold, M. R., and DeFranco, A. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4148-4152 [Abstract/Free Full Text]
25. Han, J., Lee, J. D., Tobias, P. S., and Ulevitch, R. J. (1993) J. Biol. Chem. 268, 25009-25014 [Abstract/Free Full Text]
26. Mirrington, R. N., Ritchie, E., Shoppee, C. W., and Taylor, W. C. (1964) Tetrahedron Lett. 7, 365-370 [CrossRef]
27. Nozawa, K., and Nakajima, S. (1979) J. Nat. Prod. (Lloydia) 42(4), 374-377 [CrossRef]
28. Cheng, H. C., Nishio, H., Hatase, O., Ralph, S., and Wang, J. H. (1992) J. Biol. Chem. 267, 9248-9256 [Abstract/Free Full Text]
29. Chandler, D. B., and Fulmer, J. D. (1987) J. Immunol. 139, 893-898 [Abstract]
30. Mitchell, J. A., Akarasereenont, P., Thiemerman, C., Flower, R., and Vane, J. R. (1993) Proc. Natl. Acad. Sci. (U. S. A.) 90, 11693-11697 [Abstract/Free Full Text]
31. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [Medline] [Order article via Infotrieve]
32. Blantz, R. C., and Wilson, C. B. (1976) J. Clin. Invest. 58, 899-911
33. Feng, L., Xia, Y., Tang, W. W., Hwang, D., and Wilson, C. (1993) J. Am. Soc. Nephrol. 4(3), 452 (abstr.)
34. Powell, W. S. (1982) Methods Enzymol. 86, 467-477 [Medline] [Order article via Infotrieve]
35. Bolen, J. B., Rowley, R. B., Spana, C., and Tsygankov, A. Y. (1992) FASEB J. 6, 3403-3409 [Abstract]
36. Uehara, Y., Fukazawa, H., Murakami, Y., and Mizuno, S. (1989) Biochem. Biophys. Res. Commun. 163, 803-809 [CrossRef][Medline] [Order article via Infotrieve]
37. Uehara, Y., Murakami, Y., Sugimoto, Y., and Mizuno, S. (1989) Cancer Res. 49, 780-785 [Abstract/Free Full Text]
38. June, C. H., Fletcher, M. C., Ledbetter, J. A., Schieven, G. L., Siegel, J. N., Philips, A. F., and Samelson, L. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7722-7726 [Abstract/Free Full Text]
39. Glaser, K. B., Asmis, R., and Dennis, E. A. (1990) J. Biol. Chem. 265, 8658-8664 [Abstract/Free Full Text]
40. Glaser, K. B., Sung, A., Bauer, J., and Weichman, B. M. (1993) Biochem. Pharmacol. 45, 711-721 [CrossRef][Medline] [Order article via Infotrieve]

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