INTRODUCTION

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The -macroglobulins are part of a large super family including human ₂-macroglobulin (₂M)¹ (1, 2). Proteolytic attack on the bait region or direct nucleophilic attack on the thiol ester bonds of human ₂M subunits triggers a major conformational change that exposes receptor recognition sites present in each of the four ₂M subunits (2, 3). Two receptors bind ₂M*, namely, LRP/₂MR and a recently discovered ₂M signaling receptor (₂MSR) (4-13). LRP/₂MR is a scavenger receptor that binds a wide variety of ligands. Binding of ₂M* to LRP/₂MR is followed by uptake and degradation in lysosomes but not activation of a signaling cascade (7, 8, 12). By contrast, binding of ₂M* or RBF to ₂MSR triggers classical signaling cascades and regulates cell proliferation (6-14).

The agonist-induced entry of Ca²⁺ from the extracellular medium is of major importance in the cytosolic Ca²⁺ signals that link activation of various receptors on the cell surface with the initiation and control of cell functions (15-17). Elevated cytosolic Ca²⁺ modulates specific cell cycle events and DNA synthesis (18-23). Binding of ₂M* to ₂MSR raises p21^RASGTP levels 2-3-fold in macrophages and pretreatment with wortmannin, a specific inhibitor of PI3K, does not affect ₂M*-induced increases in p21^RASGTP levels (24).

Cellular 3-phosphoinositides are generated through the action of a family of PI3Ks (25, 26). PI3K activity was first reported in association with v-SRC and v-RAS oncoproteins, where it catalyzes phosphorylation of inositol at the D-3 position of phosphatidylinositol (PtdIns), PtdIns 4-phosphate, and PtdIns 4,5-bisphosphate (25-27). Several down stream protein substrates for PI3K have been identified, which include certain protein kinase C isoforms (PKC, PKC, PKC, PKC) (25-27) and the plekstrin homology domain containing protein kinases cAKT and BTK (28). An increase in the intracellular concentration of PtdIns 3,4-bisphosphate and PtdIns 3,4,5-trisphosphate is observed in several cell types on stimulation with growth factors, cytokines, insulin, f-Met-Leu-Phe, agents that activate RAS, and viral transformation (25-27). Signaling by 3-phosphoinositides regulates diverse functions such as mitogenesis, cell growth, membrane ruffling, chemotaxis, oxidant production, secretory responses, insulin-mediated membrane translocation of the glucose transporter, membrane trafficking of growth factor receptors, cell adhesion, and Na/H⁺ exchange (25-27). Since many of the cellular responses elicited upon ligation of ₂MSR with receptor-recognized forms of ₂M are similar to those elicited upon binding of growth factors to their receptors, we studied the activity of PI3K by measuring the formation of PtdIns 3,4,5- trisphosphate (PIP₃), in murine macrophages stimulated with ₂M*. Ligation of ₂MSR increases the wortmannin-sensitive formation of PIP₃ 2-3-fold in a concentration-dependent manner and that the agonist-induced formation of PIP₃ is influenced by [Ca²⁺]_i levels.

	EXPERIMENTAL PROCEDURES

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Materials-- Human ₂M, ₂M-methylamine (₂M*), RBF and its mutants K1370A and K1374R were prepared as described (13). The sources of thioglycollate-elicited macrophages and cell culture requirements have been described previously (7-10). PtdIns 4-phosphate (PIP), PtdIns 4,5-bisphosphate (PIP₂) and PtdIns 3,4,5-trisphosphate (PIP₃), were from Biomol (Plymouth Meeting, PA). Insulin, wortmannin, thapsigargin, fatty acid-free bovine serum albumin (BSA) and molybdenum blue spray were from Sigma. Fura 2/AM and BAPTA/AM were from Molecular Probes (Eugene, OR). [³H]Thymidine (specific activity 70 Ci/mmol) and [³H]myoinositol (specific activity 20 Ci/mmol) were from American Radiolabeled Chemicals (St. Louis, MO). Silica gel G plates were from Analtech (Dover, DE). All other reagents used were of analytical grade.

Measurement of PtdIns 3,4,5-Trisphosphate Formation-- PIP₃ formation in murine peritoneal macrophages was measured essentially according to the method of Okada et al. (29) except that [³H]myoinositol was used to label inositol lipids in place of ³²P_i. Briefly thioglycollate-elicited macrophages (~8 × 10⁶/well) were collected in Hanks' balanced salt solution (HHBSS) containing 10 mM HEPES, pH 7.4, and were allowed to adhere for 2 h in RPMI 1640 medium containing 2 mM glutamine, 12.5 units of penicillin/ml, and 6 µg of streptomycin/ml, and 5% fetal bovine serum at 37 °C in a humidified CO₂ (5%) incubator. Nonadherent cells were removed with cold HHBSS, and a volume of RPMI 1640 medium was added containing the additions listed above except that 0.2% fatty acid-free BSA was substituted for the serum. To each well [³H]myoinositol, 30 µCi/ml, was added, and the cells were incubated as above for 20 h. The monolayers were washed four times with the above RPMI 1640 medium, a volume of the medium added to each well, and the cells preincubated for 3 min at 37 °C before stimulation with different agonists for 10 min. In experiments where the effect of wortmannin on agonist-induced formation of PIP₃ was studied, it was incubated (30 nM) with samples for 30 min at 37 °C prior to addition of agonists. In experiments where the effects of modulation of intracellular Ca²⁺ by thapsigargin (100 nM) and BAPTA/AM (10 µM) were to be studied on PIP₃ formation, the former was added 10 min and the latter 30 min before the addition of the agonist. The reaction was terminated by aspirating the medium, a volume of chilled methanol was added to each well, and the lipids were extracted and separated on oxalate-impregnated silica gel G plates as described by Okada et al. (29). Authentic standards of PIP, PIP_2, and PIP₃ were co-chromatographed with each run. The chromatoplates were air-dried and phospholipid spots detected by lightly spraying with molydenum blue spray (30). The R_F values obtained under the experimental conditions for PIP, PIP₂, and PIP₃ were 0.63, 0.23, and 0.12, respectively. Gel areas corresponding in R_F values to PIP₃ were scraped into scintillation vials and the radioactivity determined by liquid scintillation counting. In preliminary experiments, the identity of ³H-labeled PIP, PIP_2, and PIP₃ on chromatoplates was established by 1) autoradiography of developed chromatoplates on Kodak BioMax film using BioMax TranScreen-LE intensifying screen (Eastman Kodak Co.) at 70 °C for 10 days and comparing the R_F values of radioactive spots with authentic standards co-chromatographed and (2) by adding authentic standards of PIP, PIP_2, and PIP₃ (15 µg each) to samples prior to chromatography and spraying the developed chromatoplates with molydenum blue spray (30).

Measurement of DNA Synthesis-- DNA synthesis was measured according to Charlesworth and Rozengurt (11, 23). Briefly, 2-h-adhered macrophages (4 × 10⁵ cells/well) were incubated in a volume of RPMI 1640 medium containing glutamine, penicillin, streptomycin, and 0.2% fatty acid-free BSA. To each well [³H]thymidine (2 µCi/ml) was added followed by the addition of different ligands to the respective wells and the incubations continued as above for 20 h. In experiments where the effects of wortmannin (30 nM) were examined on RBF-induced DNA synthesis, it was added 30 min before adding the ligand, and the incubations were performed as above. The incubations were terminated by aspirating the medium, a volume of 5% trichloroacetic acid was added to each well, and plates were left on ice for 30 min. Trichloroacetic acid was aspirated and cells washed once more with trichloroacetic acid followed by washing three times with cold HHBSS. The cells were lysed in 1 N NaOH and radioactivity determined by liquid scintillating. For protein measurement, identically incubated, but untreated, cells were lysed in 0.1 N NaOH and protein estimated according to Bradford (31).

Measurement of Inositol 1,4,5-Triphosphate and [Ca²⁺]_i-- IP₃ and [Ca²⁺]_i elicited upon exposure of murine peritoneal macrophages to ₂M* were measured as described (6-10). In experiments where the effects of wortmannin (30 nM) on ₂M*-induced changes in [Ca²⁺]_i and IP₃ were studied, it was added 30 min before the agonist.

	RESULTS AND DISCUSSION

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₂MSR Ligation with ₂M* Increases PIP₃ Levels-- The effect of ₂M* on the synthesis of PIP₃ in macrophages is shown in Fig. 1. The maximum synthesis of PIP₃ occurred at a ligand concentration of ~50 pM (Fig. 1A). The kinetics of PIP₃ synthesis is similar to that noted previously for p21^RASGTP formation (24) in macrophages stimulated with ₂M*. Since wortmannin treatment had no effect on ₂M*-stimulated p21^RASGTP synthesis (24), PI3K is downstream of RAS, consistent with the report that PI3K is a substrate for activated RAS (32). The synthesis of PIP₃ stimulated with 100 pM of ₂M* was maximal after a 10-min period of incubation but declined at longer periods of incubations (Fig. 1B). The ₂M*-induced synthesis of PIP₃ was comparable with the effect of insulin (20 nM) (Fig. 2A), a potent activator of PI3K (33). That the increase in PIP₃ formation occurs due to the binding of ₂M* to ₂MSR was confirmed by using a RBF of ₂M and its mutants (Fig. 2B). Both RBF and its mutant K1370R, which bind to ₂MSR and generate signaling events similar to that of ₂M* (13), caused a 2-fold increase in PIP₃ synthesis (Fig. 2B). By contrast, the binding site mutant K1374A, which binds poorly to ₂MSR, does not elicit increases in IP₃ formation or increases in [Ca²⁺]_i (13) and failed to stimulate PIP₃ synthesis (Fig. 2B). Wortmannin, a potent and specific inhibitor of PI3K activity (34), completely inhibited ₂M*-, RBF-, and insulin-induced increases in PIP₃ synthesis (Fig. 1A and 2). We also tested the effect of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002)on ₂M*-induced PIP₃ synthesis. LY294002 is a specific inhibitor PI3K, albeit its EC₅₀ is greater than wortmannin (35, 36). This inhibitor almost completely abolished PIP₃ synthesis in macrophages exposed to ₂M* (Table I).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   PIP3 formation as a function of 2M* concentration. A, effect of 2M* concentrations on PIP3 formation in the absence () and presence of () of wortmannin (30 nM, 30 min/37 °C/prior to addition of 2M*). B, effect of time of incubation with 2M* (100 pM) on PIP3 formation in peritoneal macrophages. Values are the average of duplicate experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Modulation of PIP3 synthesis by insulin, RBF, 2M*, wortmannin, and thapsigargin. A, PIP3 values are the mean ± S.E. from three individual experiments performed in duplicate. The data are for buffer (column 1), 2M* (100 pM) (column 2), insulin (20 nM) (column 3), wortmannin + 2M* (column 4), wortmannin + insulin (column 5). Wortmannin (30 nM) was added 30 min prior to stimulation with agonists. B, PIP3 synthesis in cells treated with RBF, binding site mutants K1370A and K1374R. Values are mean ± S.E. from three individual experiments performed in duplicate. The data are for buffer (column 1), RBF (100 pM) (column 2), mutant K1370A (100 pM) (column 3), mutant K1374R (100 pM) (column 4), and RBF + wortmannin (30 nM, 30 min before RBF) (column 5). C, modulation of PIP3 synthesis by intracellular Ca2+ modulators. PIP3 values are the mean ± S.E. from two individual experiments run in triplicate. The data are for buffer (column 1), 2M* (100 pM) (column 2), thapsigargin (100 nM/10 min) (column 3), thapsigargin 10 min prior to 2M* (100 pM) (column 4), and BAPTA/AM (10 µM) 30 min prior to 2M* treatment (column 5). PIP3 levels in buffer- and BAPTA/AM (10 µM)-treated cells in the absence of 2M* were 63.17 ± 0.90 and 69.02 ± 3.00, respectively, indicating that this treatment alone does not affect intracellular PIP3 levels.

View this table: [in this window] [in a new window]   Table I Effect of LY294002 on 2M*-induced PIP3 synthesis in macrophages

Wortmannin Inhibits RBF-induced DNA Synthesis-- A number of signal transduction pathways have been implicated in regulating cell growth and differentiation in response to G-protein-coupled receptor agonists that activate protein tyrosine kinase receptors (see Ref. 40 for review). These pathways include cascades involving the Ser/Thr kinase families, MAPK, and the ribosomal S6 kinases (25, 26, 28-41). PI3K has been implicated in the regulation of cell growth in a variety of cell types (25, 26, 42). The lipid product of PI3K is not broken down by phospholipase C but seems to act as second messenger playing a role in Ca²⁺ mobilization, actin arrangement, and activation of Ser/Thr kinases such as isoforms of PKC and protein kinase B (PKB also known as cAKT). The latter are activated consequent to PI3K activation in cells treated with growth factors and mitogens, overexpression of PI3K, and inhibited by wortmannin and by dominant negative subunit mutants of PI3K (25-27, 41). Downstream targets of PKB include p70 ribosomal kinase S6 involved in up-regulation of transcripts for ribosomal proteins and elongation factors (43, 44). We have assessed the involvement of PI3K in RBF-induced DNA synthesis in macrophages by using wortmannin (Fig. 3A). Incubation of cells with wortmannin (30 nM/30 min/37 °C) prior to stimulation with RBF (100 pM) nearly abolished RBF-induced DNA synthesis (Table II), which shows that the PI3K signaling pathway is involved in DNA synthesis in cells stimulated with receptor-recognized forms of ₂M. The PI3K inhibitor LY294002 also nearly abolished DNA synthesis induced by RBF or ₂M* (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of wortmannin on 2M*-induced changes in IP3 synthesis, intracellular Ca2+, and entry of extracellular Ca2+. A, effect of wortmannin on 2M*-induced IP3 formation. Values are the average of two experiments. The data are for: 2M* (100 pM) (), wortmannin (30 nM) + 2M* (), and wortmannin (30 nM) alone (). B, [Ca2+]i as a function of 2M* addition in the absence and presence of a high concentration of external Ca2+. The 2M* concentration was 100 pM (first arrow), and the Ca2+ concentration was 1 mM (second arrow). Representative changes in [Ca2+]i are shown in a single cell in the absence () and presence () of wortmannin. Three individual experiments were performed using 40-45 cells/experiment.

Chelation of [Ca²⁺]_i with BAPTA/AM Inhibits ₂M*-induced PIP₃ Synthesis-- We have previously reported the dependence of protein and DNA synthesis on intracellular Ca²⁺ levels in macrophages stimulated with ₂M* (11). We have now examined the role of [Ca²⁺]_i on PIP₃ synthesis in macrophages stimulated with ₂M* in several ways: 1) by modulating [Ca²⁺]_i with thapsigargin (100 nM/10 min/37 °C), an endoplasmic reticulum Ca²⁺-ATPase inhibitor that raises [Ca²⁺]_i by releasing Ca²⁺ from both IP₃-dependent and IP₃-independent internal Ca²⁺ pools and 2) by use of BAPTA/AM (10 mM/30 min/37 °C) that chelates [Ca²⁺]_i. Thapsigargin alone increased PIP₃ synthesis comparable with that seen with ₂M* or with thapsigargin plus ₂M* (Fig. 2C). By contrast, BAPT/AM nearly abolished ₂M*-induced PIP₃ synthesis (Fig. 2C). We have reported previously that manipulating IP₃ and [Ca²⁺]_i profoundly alters agonist-induced increases in protein and DNA synthesis (10, 11). In light of the importance of [Ca²⁺]_i in ₂M-induced DNA synthesis, and inhibition of DNA synthesis by wortmannin (Fig. 2C) we evaluated the effect of wortmannin on ₂M*-induced synthesis of IP₃ and changes in [Ca²⁺]_i (Fig. 3). Wortmannin by itself showed no effect on IP₃ synthesis in macrophages, and when administered before ₂M*, it only slightly attenuated IP₃ synthesis (about 10-15%) compared with ₂M*-treated cells (Fig. 3A). As expected, treatment of cells with wortmannin before ₂M* only slightly attenuated both the IP₃-induced increase in [Ca²⁺]_i as well as Ca²⁺ entry from the medium (Fig. 3B).