Wortmannin inhibits mitogen-activated protein kinase activation by platelet-activating factor through a mechanism independent of p85/p110-type phosphatidylinositol 3-kinase.

We have shown previously that wortmannin partially inhibits mitogen-activated protein kinase (MAPK) activated by platelet-activating factor (PAF) in guinea pig neutrophils (Ferby, M. I., Waga, I., Sakanaka, C., Kume, K., and Shimizu, T.(1994) J. Biol. Chem. 269, 30485-30488). To identify whether p85-dependent phosphatidylinositol 3-kinase is a target molecule of wortmannin in this inhibitory process, we established a murine macrophage cell line (P388D1), inducibly expressing a dominant-negative p85, Δp85. Upon induction of Δp85 by isopropyl-β-D-thiogalactopyranoside, PAF still induced unaltered activation of MAPK, which was inhibited completely by wortmannin and 1,2-bis-(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester in an additive manner. Thus, PAF activates MAPK in P388D1 cells via two distinct pathways, one calcium-dependent and another calcium-independent, but wortmannin-sensitive. The inhibition of calcium-independent activation of MAPK by wortmannin does not involve p85-dependent phosphatidylinositol 3-kinase.

The phospholipid mediator platelet-activating factor (PAF) 1 acts through a heterotrimeric G-protein-coupled receptor to mediate divergent biological activities in a wide variety of blood cells such as platelets, neutrophils, macrophages, eosinophils, and lymphocytes (1-7), thus making it a remarkably versatile mediator of inflammation and immune responses (8,9). PAF triggers various early signaling events, including activation of phospholipases C, D, and A 2 (10 -12), as well as phosphatidylinositol 3-kinase (PI 3-K) (13,14). Furthermore, PAF has been shown to activate mitogen-activated protein kinase (MAPK) in human platelets (15), human B cell lines (16), guinea pig neutrophils (17), and CHO cells expressing the cloned PAF receptor (18). MAPK is a widely distributed serine-threonine kinase considered to be a key mediator of mainly proliferative and mitogenic responses through regulating activities of several transcription factors (20) and cytosolic PLA 2 etc. (21,22).
PAF has been shown recently to activate PI 3-K in neutrophils (13) and human B cells (23). PI 3-K is a phospholipid kinase that has received much attention lately since its main physiological product, phosphatidylinositol (3,4,5)-trisphosphate, appears to be a second messenger involved in membrane ruffling, superoxide generation in neutrophils, glucose transport control in adipocytes, and neurite outgrowth on PC12 cells (24 -26). Growth factor receptors, cytokine receptors, and Gprotein-coupled receptors are capable of stimulating PI 3-K activity (27)(28)(29). The PI 3-K pathway has been fairly well described in growth factor signaling, much thanks to the molecular cloning of PI 3-K activated by platelet-derived growth factor (30,31). This enzyme consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. Enzyme activity requires association of the regulatory and catalytic subunits and by disrupting the binding site for p110 on the p85 subunit, PI 3-K activity is lost (32). The characterization of G-proteinmediated PI 3-K activity is currently unclear and has been proposed to involve both heterodimeric p85-dependent PI 3-K (46) and a distinct form of PI 3-K regulated by the ␤␥-subunit of heterotrimeric G-proteins (33,34).
Wortmannin is a fungal metabolite that blocks many functional responses in neutrophils and has been characterized as an inhibitor of PI 3-K at nanomolar order (35)(36)(37) and myosin light chain kinase (MLCK) at micromolar order (38). Recent studies in our laboratory and others have demonstrated that wortmannin partially inhibits MAPK activation caused by PAF in guinea pig neutrophils (17) and CHO cells 2 or vasopressin (V1)-induced MAPK activation in rat 3Y1 cells (19). In these cells, wortmannin in combination with the use of calcium chelator or down-regulation of calcium-dependent protein kinase C completely inhibited the ligand-induced MAPK activation in an additive manner. Therefore, it was speculated that wortmannin targets on a molecule(s) involved in a calcium-independent and protein kinase C-independent pathway. The present study was undertaken to identify whether a p85-dependent PI 3-K is the target of wortmannin involved in PAF-induced activation of MAPK. By inducibly expressing dominant-negative p85, ⌬p85, in the macrophage cell line, P388D1, we here demonstrate that wortmannin inhibits MAPK activation by PAF through a mechanism independent of the conventional p85/p110 heterodimeric PI 3-K.
Cell Culture and Cell Preparation-P388D1 cells were grown in RPMI 1640 medium containing 10% fetal bovine serum (heat-inactivated) at 37°C in a humidified atmosphere (5% CO 2 ) to ϳ70% confluence before passage (or alternatively experimental use). Adherent cells were recovered by scraping with a silicon cell scraper. Experiments were done on serum-starved (12-16 h) cells suspended in Hanks' balanced salt solution (HBSS) supplemented with 10 mM Hepes-NaOH (pH 7.4) and 2 mg/ml BSA. Guinea pig peritoneal neutrophils were harvested and prepared as described (35).
Inducible Expression of ⌬p85 in P388D1-Bovine p85␣ with a deletion of 34 amino acids in the p110 binding region from 479 -513 and insertion of other amino acids (Ser-Arg) in this deleted region (32,39) were subcloned into the expression vector pOPRSVICAT (pOPRSVI-⌬p85). This vector contains a geneticin resistance gene and Lac operator sequences that control expression of the downstream gene by IPTG. First, cells were transfected by electroporation with the lac repressor expressing vector p3ЈSS containing a hygromycin resistance gene. Transformants were selected by maintaining cells in medium supplemented with 200 g/ml hygromycin B and isolated with iron cylinders and examined for Lac repressor expression by Northern blotting. Two clones exhibiting the highest Lac repressor expression were transfected by electroporation with the pOPRSVI-⌬p85 plasmid, and the Neo-resistant clones were selected by growth in 600 g/ml geneticin. Clones were examined for IPTG-induced p85 expression by enzyme-linked immunosorbent assay (ELISA) method (see below). Three clones consistently displaying high IPTG-induced p85 expression were selected for further experiments, denoted ⌬p85a, ⌬p85b, and ⌬p85c. After selection, clones were maintained in medium supplemented with 400 g/ml geneticin and 150 g/ml hygromycin B.
ELISA for Quantitation of p85 Expression-Basically performed by standard procedures (40). Ninety-six-well assay plates (Corning, low stringency polystyrene plates) were coated with anti-rat polyclonal p85 (PI 3-K) antibody (1/1,000 dilution) in phosphate-buffered saline at 4°C overnight, rinsed well, and blocked with 20% fetal bovine serum in phosphate-buffered saline overnight. After a mild rinse, cell lysates (50 l/well) were applied and incubated for 2 h at room temperature. Plates were rinsed three times with Tris-buffered saline (TBS) containing 0.05% Tween 20. Anti-mouse monoclonal p85 antibody (1/1,000 dilution) was added to the wells and left for 2 h at room temperature. After several rinses with TBS containing 0.05% Tween 20, wells were incubated with peroxidase-labeled anti-mouse IgG antisera (1/3,000 dilution) for 2 h at room temperature, followed by extensive washing in TBS and measurement of peroxidase activity.
Depletion of Intracellular Ca 2ϩ -P388D1 cells (5 ϫ 10 6 /ml) were suspended in Ca 2ϩ -free HBSS supplemented with 10 mM Hepes-NaOH (pH 7.4) and 2 mg/ml BSA and were incubated for 30 min at 25°C in the presence of 20 M BAPTA/AM, washed twice, and resuspended in the same buffer without BAPTA/AM (17).
Measurement of Intracellular Ca 2ϩ Concentrations-Cells suspended in Ca 2ϩ -containing HBSS were loaded with 4 M Fura-2/AM at room temperature for 20 min, washed twice, resuspended in HBSS to a concentration of 10 6 cells/ml, and measured for intracellular Ca 2ϩ , as described (17,41). After a 5-min equilibration at 37°C under gentle stirring, ligand was added, and elevations of intracellular Ca 2ϩ concentrations were measured on a spectrofluorometer (model CAF-100 or CAM-230, JASCO, Tokyo), with emission wavelength set at 510 nm and excitation wavelengths pendeling between 340 and 380 nm.
Superoxide Production and ␤-Glucuronidase Release-Superoxide released from cells into the surrounding medium was detected as reduction of horse heart cytochrome c as described (35,42), and released ␤-glucuronidase activity was measured as described (42).

Differential Effects of Wortmannin on MAPK and PI 3-K Activation-
We have shown previously that wortmannin partially inhibits MAPK activated by PAF in guinea pig neutrophils (17). First, we compared the potency by which wortmannin inhibits MAPK, PI 3-K, and various biological functional responses. Pretreatment of guinea pig neutrophils with various doses of wortmannin for 10 min at 37°C, dose-dependently inhibited PAF (100 nM)-induced activation of MAPK and PI 3-K, production of superoxide, and release of ␤-glucuronidase (Fig. 1). While wortmannin inhibited PI 3-K activation, enzyme release and superoxide production with similar IC 50 values varying from 5 to 20 nM, doses around 300 nM were required for half-maximal inhibition of MAPK activation. The discrepancy , and production of superoxide (q) was measured on guinea pig peritoneal neutrophils, pretreated with various doses of wortmannin for 10 min at 37°C, prior to stimulation with 100 nM PAF. Samples for MAPK assay were stimulated for 1 min (100% activation correspond to a 5.2-fold increase over basal 1.1 ϫ 10 4 cpm). Cells were stimulated for 30 s, and the samples were assayed for in vivo PI 3-K activity (100% activation correspond to a 2.7-fold increase in phosphatidyl (3,4,5)-trisphosphate production). Cells (5 ϫ 10 6 /ml) measured for superoxide production and ␤-glucuronidase release assays were stimulated for 2 min before extracellular medium was recovered and subjected to measurements. 100% activation of superoxide production correspond to a 5.6-fold over basal (5 nM superoxide) and for ␤-glucuronidase release to a 2.9-fold increase. See "Experimental Procedures" for assay procedures. Symbols and vertical bars represent the mean and S.D. of three experiments. p85/PI 3-K-independent Inhibition of MAPK by Wortmannin raised a possibility that PI 3-K, in particular a conventional isoform, p85-dependent PI 3-K, is not a target of wortmannin in the inhibitory process of PAF-induced MAPK activation. The following experiments were done, therefore, to determine at the molecular level whether p85-dependent PI 3-K is a target of wortmannin effect.
Activation of MAPK in P388D1 Cells and Inhibition by Wortmannin-Murine macrophage-like P388D1 cells were examined for activation of MAPK, following addition of 100 nM PAF. PAF evoked a rapid and transient activation that peaked 1 min after stimulation, with a 2-3-fold activation of MAPK ( Fig. 2A). Pretreatment of cells with 1 M wortmannin for 10 min at 37°C resulted in a partial (50 -60%) inhibition of MAPK activation in response to PAF (Fig. 2B), correlating with our previous finding that wortmannin partially inhibits PAF-induced MAPK activation in guinea pig neutrophils (Ref. 17 and Fig. 1). Treatment with wortmannin alone had no effects on PAF-evoked MAPK activity (data not shown). To determine which kind of G-protein(s) coupled with MAPK and calcium mobilization, cells were treated overnight with 100 ng/ml PTX, either response was not altered (data not shown), suggesting that PAF couples with PTX-insensitive G-protein(s) in P388D1 cells.
Inducible Expression of Dominant-negative p85, ⌬p85, in P388D1 Cells-We obtained three independent P388D1 cell lines exhibiting high expression of ⌬p85 upon induction by IPTG, as measured by ELISA. IPTG (2 mM) induced a 5-15-fold increase of ⌬p85 expression over the basal level (nontransfected cells) (Fig. 3A) that reached a maximum level after 5-6 h induction time and remained unaltered for at least 24 h (data not shown). IPTG at doses higher than 2 mM did not enhance the induction level (not shown).
Wild-type and ⌬p85a cells stimulated with 100 nM PAF, or 50 ng/ml GM-CSF, were lysed, immunoprecipitated with anti-p85 antiserum, and subjected to in vitro PI 3-K assay (Fig. 3B). PAF induced only a very slight increase in the PI 3-K activity, while GM-CSF on the other hand evoked a 3-5-fold increased production of PIP. Wortmannin (500 nM) completely blocked the response by PAF or GM-CSF. In ⌬p85a cells, the basal as well as PAF-or GM-CSF-induced activities were lowered ϳ2-fold, indicating that the lac repressor system is somewhat leaky, allowing some ⌬p85 expression, even without induction with IPTG. However, in IPTG-induced ⌬p85a cells, GM-CSF totally failed to induce PIP production. Similar results were obtained for the ⌬p85b and ⌬p85c clones (data not shown).
Wild-type and ⌬p85-expressing P388D1 clones were examined for Ca 2ϩ influx elicited by PAF, as monitored by Fura-2 (Fig. 3C). PAF caused an immediate and transient, ϳ3-fold increase of intracellular Ca 2ϩ in wild-type as well as ⌬p85expressing clones induced with IPTG for 6 h, thus indicating that ⌬p85 expression and differences between the individual clone did not cause any major changes in PAF receptor-mediated calcium signaling.
Inhibition of PAF-induced MAPK Activity by Wortmannin in  1 and 6). C, increase of intracellular Ca 2ϩ induced by 100 nM PAF (arrows), as monitored by Fura-2, in wild-type and ⌬p85ac P388D1 cells.
Wild-type versus ⌬p85-transfected Cells-In the following, we examined the effect of dominant-negative p85 expression on PAF-induced MAPK activity. Preincubating cells with 500 nM to 1 M wortmannin for 10 min at 37°C inhibited MAPK activation induced by PAF (100 nM) with ϳ50% in wild-type as well as ⌬p85-expressing cells, subjected to IPTG induction (2 mM) for 6 h (Fig. 4). Although some differences were observed in magnitude of the PAF-induced response between the individual clones, all were inhibited by wortmannin to approximately the same extent. The results clearly demonstrated that ⌬p85 did not affect the same target as wortmannin involved in PAFmediated MAPK activation.
Since 1 M wortmannin only partially inhibited PAF-induced MAPK activity in wild-type and ⌬p85-expressing P388D1 clones, we next examined the effect of calcium depletion on the wortmannin-insensitive part of the response in both wild-type and ⌬p85-transfected cells. Preloading cells with BAPTA/AM under conditions that were shown previously to abolish PAFinduced calcium response in guinea pig neutrophils (17) caused a ϳ50% inhibition of the PAF-induced MAPK response in wildtype P388D1 cells. When combining BAPTA/AM loading with wortmannin treatment, a complete inhibition occurred (Fig.  5A). When the ⌬p85a clone, induced for ⌬p85 expression with IPTG for 6 h, was used in the same experiment, essentially the same result was obtained (Fig. 5B). Neither BAPTA/AM nor wortmannin use alone had effect on MAPK activity (data not shown).
Effect of MLCK Inhibitors on PAF-induced MAPK Activation-Since p85/PI 3-K apparently is not the target of wortmannin involved in PAF-induced MAPK activation, we next examined whether another reported target of wortmannin, MLCK is instead involved in PAF-mediated activation of MAPK. Preincubation of P388D1 cells with 50 M ML-7 or 100 M ML-9 for 30 min at 37°C, concentrations to specifically inhibit MLCK (43,44), did not have any significant effects on the basal nor PAF (100 nM)-induced MAPK activity (Table I).
All these results indicate that p85-dependent PI 3K is not a target of wortmannin in the inhibitory process of MAPK activation through Ca 2ϩ -independent pathway. DISCUSSION Recent studies from our laboratory and others (17,19) have demonstrated the involvement of a wortmannin-sensitive target in G-protein-mediated MAPK activation. PAF-induced MAPK activation in guinea pig neutrophils (17) and CHO cells carrying the PAF receptor 2 were partially inhibited by wortmannin, while on the other hand, wortmannin blocked completely somatostatin-induced MAPK activation in CHO cells expressing a cloned somatostatin receptor (SSTR4) (45). SSTR4 does not cause any Ca 2ϩ signaling in the cells, but potently activates MAPK (45). These findings raised a reasonable possibility that PI 3-K might be involved in G-protein-dependent MAPK activation in a Ca 2ϩ -independent pathway. In rat 3Y1 fibroblasts stimulated with vasopressin, a similar additive effect was observed with wortmannin treatment and protein kinase C down-regulation (19).
The mechanism by which G-protein-coupled receptors induce PI 3-K activation is still unclear. Partially purified PI 3-K from platelets (46) and neutrophils (33) activated by the ␤␥-subunit of G-proteins might result from a p85-p110 interaction (46) or might involve a distinct G-protein-specific PI 3-K isotype (33). The present study was, therefore, undertaken to determine whether or not p85-dependent PI 3-K is actually involved in the activation process of MAPK. A more than one order of discrepancy in the dose required to inhibit PAF-induced MAPK activation and PI 3-K is observed in guinea pig neutrophils (Fig. 1). Inhibition by wortmannin of common neutrophil functional responses appears to follow the dose dependence for inhibition of PI 3-K. The discrepancy raises the possibility that a molecule other than PI 3-K is involved in PAF-induced MAPK activation.
Among a panel of myeloid cell lines, the murine P388D1 macrophage-like cell line displayed a relatively strong (2-3fold) activation of MAPK by PAF (Fig. 2) and was thus chosen for further studies. We established three independent P388D1 transfectants, inducibly expressing a dominant-negative mu-2 C. Sakanaka and T. Shimizu, unpublished observation.  p85/PI 3-K-independent Inhibition of MAPK by Wortmannin tant of p85, ⌬p85. Although, these clones upon induction exhibited unaltered PAF-induced Ca 2ϩ response, they failed to activate PI 3-K toward PAF or GM-CSF (positive control) (Fig. 3).
Induced dominant-negative p85 expression did not affect the PAF-induced activation of MAPK (Fig. 4). Despite ⌬p85 expression, wortmannin was still inhibitory to MAPK activation by PAF in all three independent ⌬p85 clones approximately to the same extent. Furthermore, Ca 2ϩ depletion by BAPTA/AM loading partially (ϳ50%) inhibited PAF-induced MAPK activation, while combined treatment with BAPTA/AM and wortmannin completely abolished the activity, in wild-type as well as in a ⌬p85-expressing clone (Fig. 5). Neither BAPTA/AM alone nor wortmannin alone has effect on MAPK activity. These results indicate that wortmannin inhibits a target involved in a Ca 2ϩindependent pathway and further supports that p85-dependent PI 3-K is not involved in neither Ca 2ϩ -dependent nor -independent activation of MAPK by PAF.
Although characterized as a Ca 2ϩ -independent enzyme, an alternative possible target of wortmannin was MLCK, shown to be inhibited by higher doses of wortmannin, correlating with the doses required to inhibit the PAF-induced MAPK response. However, the structurally related MLCK inhibitors ML-7 and ML-9 did not affect PAF-induced MAPK activation (Table I).
In conclusion, wortmannin inhibits MAPK activation by PAF, on a target(s) other than p85-dependent PI 3-K or MLCK in P388D1 cells. One remaining possibility is that a distinct G-protein-activated PI 3-K isotype is the true target of wortmannin involved. Indeed, a PI 3-K activated by the ␣and ␤␥-subunits of G-proteins, p110␥, activated independently of p85 has most recently been cloned (34). Future investigation will have to reveal whether this or a similar enzyme is the true target in G-protein-dependent, but calcium-protein kinase Cindependent, pathway of MAPK activation.