(Received for publication, November 14, 1995; and in revised form, March 21, 1996)
From the
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, p85. Upon induction of
p85 by
isopropyl-
-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) ()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, 2, 3, 4, 5, 6, 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
(10, 11, 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
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, 25, 26) . Growth factor receptors,
cytokine receptors, and G-protein-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-protein-mediated 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 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.
Figure 1:
Dose-dependent
inhibition by wortmannin of MAPK, PI 3-K, and functional responses in
PAF-stimulated guinea pig neutrophils. MAPK () and in vivo PI 3-K (
) activity, release of
-glucuronidase (
),
and production of superoxide (
) 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
10
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
10
/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.
Figure 2:
Activation of MAPK by PAF and inhibition
by wortmannin in P388D1 cells. A, P388D1 cells (2
10
cells/ml) were challenged with 100 nM PAF for
indicated times and MAPK activity measured on cell lysates as described
under ``Experimental Procedures.'' Basal radioactivity was 9
10
cpm. B, cells were preincubated with 1
µM wortmannin or vehicle for 10 min at 37 °C, prior to
stimulation. Column 1, nonstimulated cells (1.1
10
cpm); column 2, cells stimulated with 100
nM PAF for 1 min; column 3, wortmannin-pretreated
cells stimulated with PAF. Filled circles/columns and vertical bars denote the mean and S.D., respectively, of three
experiments.
Figure 3:
Inducible expression of dominant-negative
p85 (p85) in P388D1 cells. A, wild-type and
p85a-c cells (
70% confluence in 24-well plates) were
incubated for 6 h in medium in the absence (black columns) or
presence (shaded columns) of 2 mM IPTG, medium
removed, cells lysed in 200 µl of lysis buffer/well, and subjected
to p85-ELISA as described under ``Experimental Procedures.''
p85 expression is expressed as peroxidase activity (absorbance at 450
nm); B, cell aliquots (2
10
cells) of
wild-type (lanes 1-5) and
p85a (lanes
6-10) P388D1 clones were challenged with ligand, lysed,
immunoprecipitated with polyclonal p85 antibody, and subjected to in vitro PI 3-K assay as described under ``Experimental
Procedures.'' Lanes 1 and 6, nonstimulated
cells; lanes 2 and 7, cells stimulated with 500
nM PAF for 1 min; lanes 4, 5, and 8-10, cells stimulated with 50 ng/ml GM-CSF for 3 min; lane 5,
GM-CSF-stimulated cells pretreated with 1 µM wortmannin
for 10 min; lane 9, GM-CSF-stimulated cells pretreated with 2
mM IPTG for 6 h. Experiments are repeated three times, and
means (column) ± S.D. are shown. *, p < 0.05;**, p < 0.01 as compared with the basal level (lanes 1 and 6). C, increase of intracellular
Ca
induced by 100 nM PAF (arrows),
as monitored by Fura-2, in wild-type and
p85a-c P388D1
cells.
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
2-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
influx elicited by PAF, as
monitored by Fura-2 (Fig. 3C). PAF caused an immediate
and transient,
3-fold increase of intracellular Ca
in wild-type as well as
p85-expressing 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.
Figure 4:
PAF-induced MAPK activity and its
inhibition by wortmannin in wild-type and p85a-c. MAPK
activity induced by 100 nM PAF was measured in wild-type and
IPTG-treated
p85a-c clones as described under
``Experimental Procedures.'' Black columns represent
nonstimulated samples; hatched columns, PAF-stimulated
samples; and shaded columns, PAF-stimulated samples
preincubated 10 min with 1 µM wortmannin. The basal
activities of wild-type cells were 8,407 ± 129 cpm; for
p85a, 7,230 ± 1,820 cpm; for
p85b, 9,208 ± 239
cpm; and for
p85c, 6,642 ± 582 cpm. Columns and vertical bars denote the mean and S.D., respectively, of three
experiments.
Since
1 µM 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 PAF-induced calcium response in guinea pig
neutrophils (17) caused a
50% inhibition of the
PAF-induced MAPK response in wild-type 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).
Figure 5:
Additive inhibition by BAPTA/AM and
wortmannin of PAF-induced MAPK activation. Wild-type (lanes
1-4) and p85a (lanes 5-8) cells were
treated with or without 20 µM BAPTA/AM for 30 min at room
temperature and/or 1 µM wortmannin for 10 min at 37
°C. Columns 1 and 5, control cells; column 2 and 6, cells stimulated with 100 nM PAF for 1
min; column 3 and 7, BAPTA/AM-pretreated cells; column 4 and 8, pretreatment with both BAPTA/AM and
wortmannin. Basal activity for wild-type and
p85a samples were
3,706 ± 450 cpm and 5,178 ± 354 cpm, respectively.
Neither BAPTA/AM alone nor wortmannin alone has effect on MAPK
activity. Columns and vertical bars denote the mean
and S.D., respectively, of three
experiments.
All these results indicate that p85-dependent PI 3K
is not a target of wortmannin in the inhibitory process of MAPK
activation through Ca-independent pathway.
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 ()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
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
-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
(2-3-fold) 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 mutant of p85,
p85. Although, these clones upon induction exhibited unaltered
PAF-induced Ca
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
depletion by BAPTA/AM loading
partially (
50%) 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
-independent pathway and
further supports that p85-dependent PI 3-K is not involved in neither
Ca
-dependent nor -independent activation of MAPK by
PAF.
Although characterized as a Ca-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 1).
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
C-independent, pathway of MAPK activation.