(Received for publication, June 8, 1995; and in revised form, September 4, 1995)
From the
Cytosolic phospholipase A (cPLA
) is
activated by a wide variety of stimuli to release arachidonic acid, the
precursor of the potent inflammatory mediators prostaglandin and
leukotriene. Specifically, cPLA
releases arachidonic acid
in response to agents that increase intracellular Ca
. In vitro data have suggested that these agents induce a
translocation of cPLA
from the cytosol to the cell
membrane, where its substrate is localized. Here, we use
immunofluorescence to visualize the translocation of cPLA
to distinct cellular membranes. In Chinese hamster ovary cells
that stably overexpress cPLA
, this enzyme translocates to
the nuclear envelope upon stimulation with the calcium ionophore
A23187. The pattern of staining observed in the cytoplasm suggests that
cPLA
also translocates to the endoplasmic reticulum. We
find no evidence for cPLA
localization to the plasma
membrane. Translocation of cPLA
is dependent on the
calcium-dependent phospholipid binding domain, as a calcium-dependent
phospholipid binding deletion mutant of cPLA
(
CII)
fails to translocate in response to Ca
. In contrast,
cPLA
mutated at Ser-505, the site of mitogen-activated
protein kinase phosphorylation, translocates normally. This
observation, combined with the observed phosphorylation of
CII,
establishes that translocation and phosphorylation function
independently to regulate cPLA
. The effect of these
mutations on cPLA
translocation was confirmed by
subcellular fractionation. Each of these mutations abolished the
ability of cPLA
to release arachidonic acid, establishing
that cPLA
-mediated arachidonic acid release is strongly
dependent on both phosphorylation and translocation. These data help to
clarify the mechanisms by which cPLA
is regulated in intact
cells and establish the nuclear envelope and endoplasmic reticulum as
primary sites for the liberation of arachidonic acid in the cell.
The 85-kDa cytosolic phospholipase A
(cPLA
), (
)which selectively releases arachidonic
acid from the sn-2 position of membrane phospholipids, is
crucial to the initiation of the inflammatory response. cPLA
activity is stimulated by a wide variety of agents, including the
proinflammatory cytokines interleukin 1 (1, 2) and
tumor necrosis factor(3) , macrophage colony-stimulating
factor(4) , thrombin(5, 6) , ATP(5) ,
mitogens(7, 8, 9, 10) , and
endothelin(11) . The release of arachidonic acid is the
rate-limiting step in the generation of prostaglandins and
leukotrienes, the proinflammatory eicosanoids. Cleavage of
arachidonoyl-containing phospholipids also results in the release of
lysophospholipid, the precursor of the inflammatory mediator
platelet-activating factor(12) .
cPLA is
expressed in many cell types. Many of these are associated with the
inflammatory response, such as monocytes (4) ,
neutrophils(13) , and synovial fibroblasts(14) .
However, cPLA
is also expressed in kidney, spleen, heart,
lung, liver, testis, and hippocampus(15) . This diverse pattern
of expression is consistent with accumulating evidence that in addition
to its role in inflammation, cPLA
participates in signaling
in processes such as platelet activation(6, 16) ,
tumor necrosis factor-induced cytotoxicity(17) , and cell
proliferation(8, 10, 18) .
cPLA activity is regulated both transcriptionally and
post-translationally. Post-translational activation is thought to occur
by two mechanisms. One mechanism involves agonist-induced MAP kinase
phosphorylation of cPLA
, resulting in stimulation of its
intrinsic enzymatic activity(19, 20) . The second
involves a Ca
-dependent translocation of cPLA
from the soluble to the membrane fraction of
cells(21, 22, 23) , allowing cPLA
access to its arachidonoyl-containing phospholipid substrate. As
discussed below, the results presented in this study establish that
both mechanisms are important for the stimulation of
cPLA
-induced arachidonic acid release.
In vitro studies have strongly suggested that the membrane binding function
of cPLA resides in its Ca
-dependent
phospholipid binding (CaLB) domain(22, 24) , a region
similar to the CII domain of protein kinase C. This domain has been
shown to be necessary and sufficient for Ca
-dependent
membrane binding(22, 24) . In this study, we
characterize the translocation of cPLA
in intact cells and
show that this enzyme translocates to the nuclear envelope and
endoplasmic reticulum. Deleting the CaLB domain abolishes the ability
of cPLA
to translocate to these membranes, whereas a
mutation at the MAP kinase phosphorylation site has no effect on
translocation. Each of these mutations is shown to prevent
cPLA
-induced arachidonic acid release. Our present data,
together with recent reports localizing several arachidonic
acid-metabolizing enzymes to the nuclear envelope and endoplasmic
reticulum(25, 26, 27, 28, 29) ,
establish these membranes as major sites of arachidonic acid production
and metabolism in the cell.
Interestingly,
immunofluorescent staining of cPLA in A23187-treated
E5-4 cells was consistently more intense than that seen in
untreated cells (quantitated in Fig. 1B). This is most
likely due to a greater loss of soluble cPLA
than
membrane-bound cPLA
throughout the staining procedure. A
similar interpretation was offered for the results observed upon
immunogold labeling of the arachidonic acid-metabolizing enzyme
5-lipoxygenase, which also translocates to the nuclear envelope in
response to A23187(27) . In that study, 5-lipoxygenase was
undetectable in unstimulated cells but was apparent at the nuclear
envelope after ionophore treatment. Alternatively, binding of
cPLA
to membranes may result in a better exposure of the
epitope for the monoclonal antibody, resulting in more efficient
antibody binding. In either case, the observed increase in cytoplasmic
staining intensity suggests that cPLA
translocates not only
to the nuclear envelope but also to a cytoplasmic membrane structure,
most likely the endoplasmic reticulum. Indeed, in some experiments
ionophore treatment resulted in an increase in reticular cytoplasmic
fluorescence, consistent with endoplasmic reticulum staining.
Figure 1:
Differential localization of cPLA in untreated and Ca
ionophore-stimulated cells.
CHO cells overexpressing wild type cPLA
were stimulated
with 2 µM Ca
ionophore (+A23187) for 2 min or left untreated (-A23187). A, immunofluorescence was performed
using a monoclonal antibody specific for cPLA
and a
fluorescein-conjugated second antibody. Immunofluorescent staining was
visualized by confocal microscopy (magnification, 330
). B, quantitative fluorescence imaging of cytoplasm and nuclear
envelope. The intensity of immunofluorescent staining was quantified by
measuring fluorescence before and after ionophore treatment in
equivalent areas of the cytoplasm and nuclear envelope (NE). ER+Cyto, endoplasmic reticulum +
cytoplasm.
The
plasma membrane was consistently found to be devoid of cPLA staining. To ensure that fixation had not disrupted the integrity
of the plasma membrane, CHO cells overexpressing the plasma membrane
protein E-selectin were stained. As shown in Fig. 2, E-selectin
antibody was clearly able to label the plasma membrane. No labeling of
the nuclear envelope was observed in these cells. As a control for the
specificity of the anti-cPLA
monoclonal antibody,
immunofluorescence was also performed on the parental CHO cells used
for these studies. Only faint staining was observed (Fig. 2),
similar to that seen in controls in which the monoclonal antibody was
eliminated from the staining protocol (data not shown). The lack of
endogenous cPLA
staining is likely to be due both to the
low level of cPLA
expressed in these cells and the failure
of this antibody to recognize murine (and by inference, hamster)
cPLA
efficiently. Fig. 2also shows staining of CHO
cells overexpressing PGHS-1, which metabolizes arachidonic acid to
prostaglandin. Similar staining results were obtained with a CHO line
expressing the isoform PGHS-2 (not shown). Both PGHS isoforms have been
localized to the endoplasmic reticulum and nuclear
envelope(25, 29) . As expected, neither E-selectin nor
PGHS-1 staining patterns were affected by ionophore treatment.
Figure 2:
Confocal microscopy of CHO cells
overexpressing various proteins. Indirect immunofluorescence was
performed on untransfected CHO cells and stable CHO cell lines
expressing cPLA, E-selectin, and PGHS-1, with (+) and
without(-) A23187 treatment.
Figure 3:
Translocation of cPLA is
dependent on the CaLB domain. A,
CII cells, which stably
express cPLA
lacking the CaLB domain, show similar staining
in untreated(-) and ionophore-stimulated (+) cells. Staining
and visualization are the same as described in the legend to Fig. 1. B, quantification of fluorescence intensity.
Staining intensity was measured as described in Fig. 1B.
Figure 4:
Translocation of SA505-cPLA.
Cells stably overexpressing SA505-cPLA
, which contains a
serine-to-alanine mutation at the Ser-505 MAP kinase phosphorylation
site, were probed with antibody to cPLA
. Indirect
immunofluorescence was performed as described in the legend to Fig. 1.
To confirm that deletion of
the CaLB domain, but not the mutation of Ser-505, abolished cPLA translocation, subcellular fractionation was performed using
cells expressing these mutants. After ionophore treatment, cell lysates
were centrifuged at 100,000
g, and the soluble and
particulate fractions were immunoblotted for cPLA
. As shown
in Fig. 5, both cPLA
and the SA505-cPLA
mutant redistributed to the particulate fraction upon ionophore
treatment, whereas the
CII mutant did not. The insert shown below
the
CII portion of the figure represents a longer exposure of the
blot, revealing the translocation of endogenous cPLA
in the
CII line. These data confirm that phosphorylation at Ser-505 is
not required for cPLA
translocation. Conversely,
translocation is not required for Ser-505 phosphorylation, as evidenced
by the presence of a cPLA
doublet in the
CII but not
the SA505 line. The upper band of the cPLA
doublet has
previously been established to result from phosphorylation at
Ser-505(19) . These data establish that translocation and
phosphorylation occur independently.
Figure 5:
Fractionation of lysates from wild type (WT), CII, and SA505 cells. Cells overexpressing each
cPLA
construct were stimulated with 2 µM ionophore for 10 min (+) or left untreated (-). Lysates
were spun at 100,000
g, and 5 µg of protein from
the supernatant (S) and pellet (P) fractions were
subjected to SDS-polyacrylamide gel electrophoresis. Proteins were
transferred to nitrocellulose and immunoblotted for cPLA
.
Development was by chemiluminescence. The positions of full-length
cPLA
and
CII are indicated. The panel below the
CII portion of the blot represents a longer exposure of these
lanes, demonstrating that endogenous cPLA
translocates
normally in the
CII line. The band in the
CII pellet
fractions represents a small amount of insoluble
CII. * indicates
a cross-reactive species of unknown origin.
ATP and thrombin have
previously been shown to induce arachidonic acid release in CHO cells
that overexpress cPLA(5) . However, in
fractionation experiments using these agents, we were unable to detect
the appearance of cPLA
in the pellet fraction (data not
shown). This suggests that translocation induced by ATP and thrombin is
highly transient, consistent with the transient nature of the
Ca
increase induced by these agents(33) .
Figure 6:
Effect of CII and SA505 mutations
on arachidonic acid release. Cells were labeled with
[
H]arachidonic acid for 20 h. After washing,
cells were stimulated with 2 µM A23187 (+) or left
untreated(-) for 10 min, and radioactivity was measured in the
media by scintillation counting. The levels of arachidonic acid
released from
CII (A) and SA505 (B) are compared
with parental CHO cells and E5-4.
The data presented here establish that in intact cells,
cPLA translocates to membranes in response to a rise in
intracellular Ca
. Specifically, cPLA
binds to the nuclear envelope and the endoplasmic reticulum. The
translocation of cPLA
to these sites is consistent with
previous work examining the subcellular distribution of cPLA
in Ca
ionophore-stimulated
macrophages(26) . In this study, cPLA
from
stimulated cells was detected both in the nuclear fraction and in the
membrane fraction lacking nuclei, consistent with its presence in both
the nuclear envelope and the endoplasmic reticulum. In another
study(23) , fractionation of neutrophils stimulated with
granulocyte/macrophage colony-stimulating factor and the chemotactic
peptide fMet-Leu-Phe revealed cPLA
to be in the membrane
fraction, even after nuclei were removed by centrifugation. Although
the translocation of cPLA
to the nuclear envelope could not
be addressed in this study (the presence of cPLA
in the
nuclear fraction was not examined), its presence in the membrane
fraction supports the translocation of cPLA
to the
endoplasmic reticulum. Taken together with our immunofluorescence
results, including the lack of cPLA
staining at the plasma
membrane, these data implicate both the nuclear envelope and
endoplasmic reticulum as the primary sites for arachidonic acid
production in the cell. Interestingly, PGHS-1 and
-2(25, 29) , the enzymes responsible for metabolizing
arachidonic acid to prostaglandins, are also localized at these sites.
In addition, 5-lipoxygenase, which metabolizes arachidonic acid to
leukotrienes, as well as its activating protein, FLAP, are localized to
the nuclear envelope(27) . These data suggest that arachidonic
acid is both produced and metabolized at the nuclear envelope and
endoplasmic reticulum.
In the work presented in this paper, the
localization of cPLA was examined by immunofluorescent
staining of cPLA
overexpressed in CHO cells. This approach
was chosen to assess the effects of the
CII and SA505 mutations on
cPLA
translocation. Caution must be taken with this
approach, however, as it is possible that overexpression of cPLA
may result in a wider pattern of expression than occurs
physiologically. Interestingly, cPLA
translocation to the
nuclear envelope was recently demonstrated in rat mast cells stimulated
with A23187 or IgE/antigen(34) . In this study, no obvious
staining of the endoplasmic reticulum was observed. This observation
raises the possibility that the localization of cPLA
to the
endoplasmic reticulum is a consequence of overexpression. However, it
is also possible that the relative proportion of cPLA
in
the nuclear envelope and endoplasmic reticulum varies between cell
types. Further studies will be necessary to clarify this issue. In any
case, the observed lack of plasma membrane staining, both in mast cells
and in cells overexpressing cPLA
, strongly suggests that
cPLA
does not translocate to this membrane in response to
increases in intracellular calcium.
Translocation of cPLA is abolished upon deletion of the CaLB domain but not upon
mutation of Ser-505. This suggests that translocation and
phosphorylation regulate cPLA
independently, a conclusion
supported by the observation that
CII is phosphorylated in CHO
cells (as evidenced by the doublet in Fig. 5). Both
translocation and phosphorylation are critical for A23187-induced
arachidonic acid release. The observation that these regulatory
mechanisms function independently strongly supports a model in which
phosphorylation at Ser-505 serves primarily to activate the enzymatic
activity of cPLA
(19, 20) , and
translocation allows access of the enzyme to its substrate.
This
discussion focuses on the localization of cPLA to the
nuclear envelope and endoplasmic reticulum. How cPLA
is
selectively localized to these sites is not known. The translocation of
cPLA
to the nuclear envelope is consistent with previous
data indicating that arachidonic acid is preferentially released from
the nuclear envelope, as observed in pulse-chase experiments using
[
C]arachidonate-labeled HSDM
C
cells stimulated with bradykinin(35) . Interestingly,
electron microscopic studies using [
H]arachidonic
acid have shown that the nuclear membrane is the preferred site of
initial arachidonic acid incorporation in these cells(36) .
Arachidonic acid is also incorporated rapidly into the endoplasmic
reticulum; in contrast, transit to the plasma membrane is slow. This
study shows that although arachidonic acid is preferentially
incorporated at particular sites, it becomes evenly distributed
throughout the cell. Thus, a correlation exists between the cellular
localization of cPLA
and the sites at which arachidonic
acid is initially incorporated into the cell. The significance
of this correlation, however, is not yet known.
Several arachidonic
acid-independent mechanisms could also explain the localization of
cPLA. Although Ca
can induce purified
cPLA
to bind membranes in the absence of additional
protein(24) , it remains possible that a ``docking
protein'' localized to the nuclear envelope and endoplasmic
reticulum allows more efficient binding of cPLA
at these
sites. It is unlikely that a spatially localized release of
Ca
is responsible for the targeting of
cPLA
, since our experiments revealed no translocation of
cPLA
to the plasma membrane with A23187, whose effects are
thought to include the triggering of a Ca
influx
across the plasma membrane. Another possibility is that some feature of
the plasma membrane excludes cPLA
, and cPLA
simply translocates to all cellular membranes that it is capable
of binding. Interestingly, the plasma membrane is known to be enriched
in sphingolipids compared to the nuclear envelope and endoplasmic
reticulum. It is conceivable that the increase in phospholipid packing
density that results from a high sphingolipid content may prevent
cPLA
binding. Consistent with this idea, Leslie and Channon (21) have shown that both the activity and calcium sensitivity
of cPLA
are inhibited by the increased substrate packing
density induced by sphingolipids. Clearly, a better understanding of
the lipid and protein components of distinct cellular membranes will
greatly facilitate our understanding of the mechanism(s) governing the
localization of cPLA
.
As discussed above, the
localization of cPLA is similar to that reported for PGHS-1
and -2, which metabolize arachidonic acid to
prostaglandins(25, 29) . PGHS-1 is expressed
constitutively and is thought to perform certain physiological
``housekeeping'' functions. PGHS-2, whose expression is
induced by cytokines, has been implicated in
inflammation(37, 38) . cPLA
-mediated
arachidonic acid release can couple to PGHS-2, since antisense
oligonucleotide inhibition of cPLA
decreases the level of
endotoxin-stimulated PGE
in monocytes (without affecting
PGHS-2 activity)(39) , and PGE
release in
endotoxin-stimulated monocytes has been shown to be dependent on PGHS-2
activity even in the presence of PGHS-1(40) . However, in
platelets, known from various studies to contain only PGHS-1,
inhibition of cPLA
using arachidonoyl trifluoromethyl
ketone blocks thromboxane B
formation, suggesting that
cPLA
can also couple to PGHS-1(41) . Indeed, the
association of cPLA
with the nuclear envelope and the
endoplasmic reticulum correlates well with the localization of both
PGHS-1 and PGHS-2. Although it is not yet clear which factors govern
the coupling of cPLA
with each PGHS isoform, the
colocalization of cPLA
with these enzymes and
5-lipoxygenase is certain to ensure the efficient utilization of
arachidonic acid upon agonist stimulation, consistent with a central
role for cPLA
in the agonist-induced biosynthesis of
prostaglandins and leukotrienes.