From the Department of Pathology, Duke University Medical Center,
Durham, North Carolina 27710
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INTRODUCTION |
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
p21RASGTP levels 2-3-fold in macrophages and
pretreatment with wortmannin, a specific inhibitor of PI3K, does not
affect
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 (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.
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EXPERIMENTAL PROCEDURES |
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 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
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.
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RESULTS AND DISCUSSION |
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
p21RASGTP formation (24) in macrophages stimulated
with
2M*. Since wortmannin treatment had no effect on
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
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).

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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.
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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.
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Ligation of
2MSR with results in activation of PLC
(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
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
2M. The
PI3K inhibitor LY294002 also nearly abolished DNA synthesis induced by
RBF or
2M* (data not shown).

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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.
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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).
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
2M, upon binding to the
2MSR, also induce the activation of wortmannin-sensitive PI3K. Thus as suggested earlier (10-14), in addition to being a classical endocytic receptor,
2MSR also appears to have
an additional role in tissue repair.