COMMUNICATION
Binding of Receptor-recognized Forms of alpha 2-Macroglobulin to the alpha 2-Macroglobulin Signaling Receptor Activates Phosphatidylinositol 3-Kinase*

Uma Kant Misra and Salvatore Vincent PizzoDagger

From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Ligation of the alpha 2-macroglobulin (alpha 2M) signaling receptor by receptor-recognized forms of alpha 2M (alpha 2M*) initiates mitogenesis secondary to increased intracellular Ca2+. We report here that ligation of the alpha 2M signaling receptor also causes a 1.5-2.5-fold increase in wortmannin-sensitive phosphatidylinositol 3-kinase (PI3K) activity as measured by the quantitation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 formation was alpha 2M* concentration-dependent with a maximal response at ~50 pM ligand concentration. The peak formation of PIP3 occurred at 10 min of incubation. The alpha 2M receptor binding fragment mutant K1370R which binds to the alpha 2M signaling receptor activating the signaling cascade, increased PIP3 formation by 2-fold. The mutant K1374A, which binds very poorly to the alpha 2M signaling receptor, did not cause any increase in PIP3 formation. alpha 2M*-induced DNA synthesis was inhibited by wortmannin. 1,2Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethylester a chelator of intracellular Ca2+, drastically reduced alpha 2M*-induced increases in PIP3 formation. We conclude that PI3K is involved in alpha 2M*-induced mitogenesis in macrophages and intracellular Ca2+ plays a role in PI3K activation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

The alpha -macroglobulins are part of a large super family including human alpha 2-macroglobulin (alpha 2M)1 (1, 2). Proteolytic attack on the bait region or direct nucleophilic attack on the thiol ester bonds of human alpha 2M subunits triggers a major conformational change that exposes receptor recognition sites present in each of the four alpha 2M subunits (2, 3). Two receptors bind alpha 2M*, namely, LRP/alpha 2MR and a recently discovered alpha 2M signaling receptor (alpha 2MSR) (4-13). LRP/alpha 2MR is a scavenger receptor that binds a wide variety of ligands. Binding of alpha 2M* to LRP/alpha 2MR is followed by uptake and degradation in lysosomes but not activation of a signaling cascade (7, 8, 12). By contrast, binding of alpha 2M* or RBF to alpha 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 alpha 2M* to alpha 2MSR raises p21RASGTP levels 2-3-fold in macrophages and pretreatment with wortmannin, a specific inhibitor of PI3K, does not affect alpha 2M*-induced increases in p21RASGTP 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 (PKCdelta , PKCepsilon , PKCeta , PKCxi ) (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 alpha 2MSR with receptor-recognized forms of alpha 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 alpha 2M*. Ligation of alpha 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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Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- Human alpha 2M, alpha 2M-methylamine (alpha 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 32Pi. 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, PIP2, 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 RF values obtained under the experimental conditions for PIP, PIP2, and PIP3 were 0.63, 0.23, and 0.12, respectively. Gel areas corresponding in RF 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, PIP2, 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 RF values of radioactive spots with authentic standards co-chromatographed and (2) by adding authentic standards of PIP, PIP2, 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 alpha 2M* were measured as described (6-10). In experiments where the effects of wortmannin (30 nM) on alpha 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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Abstract
Introduction
Procedures
Results & Discussion
References

alpha 2MSR Ligation with alpha 2M* Increases PIP3 Levels-- The effect of alpha 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 p21RASGTP formation (24) in macrophages stimulated with alpha 2M*. Since wortmannin treatment had no effect on alpha 2M*-stimulated p21RASGTP 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 alpha 2M* was maximal after a 10-min period of incubation but declined at longer periods of incubations (Fig. 1B). The alpha 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 alpha 2M* to alpha 2MSR was confirmed by using a RBF of alpha 2M and its mutants (Fig. 2B). Both RBF and its mutant K1370R, which bind to alpha 2MSR and generate signaling events similar to that of alpha 2M* (13), caused a 2-fold increase in PIP3 synthesis (Fig. 2B). By contrast, the binding site mutant K1374A, which binds poorly to alpha 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 alpha 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 alpha 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 alpha 2M* (Table I).


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Fig. 1.   PIP3 formation as a function of alpha 2M* concentration. A, effect of alpha 2M* concentrations on PIP3 formation in the absence (bullet ) and presence of (open circle ) of wortmannin (30 nM, 30 min/37 °C/prior to addition of alpha 2M*). B, effect of time of incubation with alpha 2M* (100 pM) on PIP3 formation in peritoneal macrophages. Values are the average of duplicate experiments.


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Fig. 2.   Modulation of PIP3 synthesis by insulin, RBF, alpha 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), alpha 2M* (100 pM) (column 2), insulin (20 nM) (column 3), wortmannin + alpha 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), alpha 2M* (100 pM) (column 2), thapsigargin (100 nM/10 min) (column 3), thapsigargin 10 min prior to alpha 2M* (100 pM) (column 4), and BAPTA/AM (10 µM) 30 min prior to alpha 2M* treatment (column 5). PIP3 levels in buffer- and BAPTA/AM (10 µM)-treated cells in the absence of alpha 2M* were 63.17 ± 0.90 and 69.02 ± 3.00, respectively, indicating that this treatment alone does not affect intracellular PIP3 levels.

                              
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Table I
Effect of LY294002 on alpha 2M*-induced PIP3 synthesis in macrophages

Ligation of alpha 2MSR with results in activation of PLCgamma (9), increases in intracellular pH (9), activation of PLA2 and PLD (37), synthesis and secretion of PAF (38) and PGE2 (39), increases in p21RASGTP levels (24), and mitogenesis (10, 11, 24). The present studies in conjunction with these previous observations show that like established tyrosine kinase receptors, cellular responses elicited upon ligation of alpha 2MSR involve several signaling cascades, including p21RAS, PI3K, and mitogen-activated protein kinase (MAPK) pathways.

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 alpha 2M. The PI3K inhibitor LY294002 also nearly abolished DNA synthesis induced by RBF or alpha 2M* (data not shown).


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Fig. 3.   Effect of wortmannin on alpha 2M*-induced changes in IP3 synthesis, intracellular Ca2+, and entry of extracellular Ca2+. A, effect of wortmannin on alpha 2M*-induced IP3 formation. Values are the average of two experiments. The data are for: alpha 2M* (100 pM) (bullet ), wortmannin (30 nM) + alpha 2M* (open circle ), and wortmannin (30 nM) alone (black-triangle). B, [Ca2+]i as a function of alpha 2M* addition in the absence and presence of a high concentration of external Ca2+. The alpha 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 (bullet ) and presence (open circle ) of wortmannin. Three individual experiments were performed using 40-45 cells/experiment.

                              
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Table II
Effect of wortmannin and RBF on DNA synthesis

Chelation of [Ca2+]i with BAPTA/AM Inhibits alpha 2M*-induced PIP3 Synthesis-- We have previously reported the dependence of protein and DNA synthesis on intracellular Ca2+ levels in macrophages stimulated with alpha 2M* (11). We have now examined the role of [Ca2+]i on PIP3 synthesis in macrophages stimulated with alpha 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 alpha 2M* or with thapsigargin plus alpha 2M* (Fig. 2C). By contrast, BAPT/AM nearly abolished alpha 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 alpha 2M-induced DNA synthesis, and inhibition of DNA synthesis by wortmannin (Fig. 2C) we evaluated the effect of wortmannin on alpha 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 alpha 2M*, it only slightly attenuated IP3 synthesis (about 10-15%) compared with alpha 2M*-treated cells (Fig. 3A). As expected, treatment of cells with wortmannin before alpha 2M* only slightly attenuated both the IP3-induced increase in [Ca2+]i as well as Ca2+ entry from the medium (Fig. 3B).

The tyrosine kinase class of receptors, which include growth factor receptors, upon activation, induce mitogenesis via a series of downstream steps that may show cellular variance and include signaling proteins Grb2, Sos, Ras, Raf, MEK, and MAPK (40). We show here, for the first time, that like insulin and other growth factors, receptor-recognized forms of alpha 2M, upon binding to the alpha 2MSR, also induce the activation of wortmannin-sensitive PI3K. Thus as suggested earlier (10-14), in addition to being a classical endocytic receptor, alpha 2MSR also appears to have an additional role in tissue repair.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-3528; Fax: 919- 684-8689; E-mail: pizzo001{at}mc.duke.edu.

1 The abbreviations used are: alpha 2M, alpha 2-macroglobulin; alpha 2M*, alpha 2M activated by proteinase or methylamine; alpha 2MSR, alpha 2M signaling receptor; LRP/alpha 2MR, low density lipoprotein receptor-related protein/alpha 2M receptor; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethylester; [Ca2+]i, intracellular free Ca2+; Fura-2/AM, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl-2)-2'-amino-5-methyl-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester; HHBSS, Hanks' balanced salt solution containing 10 mM HEPES and 3.5 mM NaHCO3; IP3, inositol 1,4,5-triphosphate; PtdIns, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5- triphosphate; PI3K, phosphatidylinositol 3-kinase; RBF, receptor binding factor; BSA, bovine serum albumin; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; MAPK, mitogen-activated protein kinase; PKC, protein kinase C.

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Abstract
Introduction
Procedures
Results & Discussion
References

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