1 Division of Experimental Diabetes and Aging, Mount Sinai School of Medicine, New York, New York 10029; and 2 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Accumulating evidence
suggests that the cytosolic calcium-independent phospholipase
A2 (iPLA2) manifests a signaling role in
insulin-secreting (INS-1)
-cells. Earlier, we reported that insulin-secretory responses to cAMP-elevating agents are amplified in
iPLA2
-overexpressing INS-1 cells (Ma Z, Ramanadham S,
Bohrer A, Wohltmann M, Zhang S, and Turk J. J Biol Chem
276: 13198-13208, 2001). Here, immunofluorescence, immunoaffinity,
and enzymatic activity analyses are used to examine distribution of
iPLA2
in stimulated INS-1 cells in greater detail.
Overexpression of iPLA2
in INS-1 cells leads to
increased accumulation of iPLA2
in the nuclear fraction.
Increasing glucose concentrations alone results in modest increases in
insulin secretion, relative to parental cells, and in nuclear
accumulation of the iPLA2
protein. In contrast, cAMP-elevating agents induce robust increases in insulin secretion and
in time-dependent nuclear accumulation of iPLA2
fluorescence, which is reflected by increases in nuclear
iPLA2
protein content and specific enzymatic activity.
The stimulated effects are significantly attenuated in the presence of
cell-permeable inhibitors of protein phosphorylation and glycosylation.
These findings suggest that conditions that amplify insulin secretion
promote translocation of
-cell iPLA2
to the nuclei,
where it may serve a crucial signaling role.
immunofluorescence; immunoaffinity; enzymatic activity; insulin secretion; nuclear localization
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INTRODUCTION |
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PHOSPHOLIPASES
A2 (PLA2) are a diverse group of enzymes
that catalyze the hydrolysis of the sn-2 substituent from
glycerophospholipid substrates to yield a free fatty acid and a
2-lysophospholipid (9, 12). Among the PLA2s is
an 85-kDa cytosolic PLA2 that does not require
Ca2+ for catalysis. This PLA2 has now been
cloned from several sources (3, 23, 44), including rat and
human pancreatic islet -cells (23, 25), and has been
proposed to be designated as the
-isoform (26, 43) of
Group VIA calcium-independent PLA2, or iPLA2
(22, 26). Several potential functions for
iPLA2
have been proposed, including a housekeeping role
(4) in phospholipid remodeling and a signaling role in
secretion (22, 23, 29).
Because glucose-stimulated insulin secretion is associated with
increased hydrolysis of the sn-2 substituent arachidonate from -cell membrane phospholipids and its accumulation within the
-cells (45, 48), we initially investigated the possible participation of iPLA2
in the insulin-secretory pathway.
Those studies (34, 37), performed in insulinoma cells and
native pancreatic islets, revealed that inhibition of
iPLA2
by the bromoenol lactone (BEL) suicide inhibitor
of iPLA2
suppresses both glucose-stimulated arachidonate
stimulation and insulin secretion. These findings raise the possibility
that iPLA2
serves a signaling role in
-cells.
Subsequent findings in studies with murine P388D1 macrophage-like cells
have led to the proposal that the iPLA2 enzyme serves a
housekeeping role in phospholipid remodeling that involves generation of lysophospholipid acceptors for incorporation of arachidonic acid
into phospholipids (4). However, detailed examination of
this process by our group (35) indicated that inhibition of the iPLA2
enzyme does not influence incorporation of
arachidonate or phospholipid remodeling in pancreatic islets or
insulinoma cells but does inhibit secretagogue-stimulated insulin secretion.
The potential signaling role of iPLA2 was further
addressed in a recently reported study (22), in which the
consequence of overexpressing iPLA2
on insulin secretion
from INS-1 insulinoma cells and of phospholipid remodeling in these
cells was examined. INS-1 cells after prolonged passaging become less
responsive to glucose-stimulated insulin secretion (36).
Late passage cells were transfected with iPLA2
cDNA, and
the iPLA2
-overexpressing INS-1 cells were subsequently
incubated with glucose, either alone or in combination with
cAMP-elevating agents. In contrast to the parental INS-1 cells, which
were unresponsive to glucose and only mildly responsive to
cAMP-elevating agents, a mild improvement in glucose responsiveness but
robust responses to IBMX and forskolin were evident in the
iPLA2
-overexpressing INS-1 cells (22). Improvements in the insulin-secretory responses, however, were not
associated with alterations in arachidonic acid incorporation into
phospholipids of the transfected cells. Collectively, these findings
reaffirm a signaling role for iPLA2
in insulin-secreting
-cells.
An intriguing finding in the study just described (22) was
that stimulation of iPLA2-overexpressing INS-1 cells
with cAMP-elevating agents was associated with increased accumulation
of iPLA2
in the nuclear region (22). In the
present study, this phenomenon is further examined by use of
immunocytofluorescence, immunoaffinity, and enzymatic activity
analyses, and contributions of possible posttranslational modifications
of the enzyme are addressed.
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METHODS |
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Materials.
INS-1 cells were generously provided by Dr. C. Newgard (University of
Texas Southwestern Center for Diabetes Research, Dallas, TX). Other
materials were obtained from the following sources: {[-32P]ATP,
(16:0/[14C]18:2)-glycerophosphocholine
[1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine
(PLPC), 55 mCi/mmol]} rainbow molecular mass standards, and enhanced
chemiluminescence (ECL) reagent from Amersham (Arlington Heights, IL);
H-89 from Biomol Research Laboratories (Plymouth Meeting, PA); SDS-PAGE supplies from Bio-Rad (Richmond, CA); pentex fraction V fatty acid-free
BSA from ICN Biochemicals (Aurora, OH); normal goat serum and
Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) from Jackson
Immuno Research Laboratories (West Grove, PA); Enlightning solution,
Slow Fade light antifade kit from Molecular Probes (Eugene, OR);
[3H]AMP (25 Ci/mmol), UDP [3H]galactose (48 Ci/mmol) from New England Nuclear (Boston, MA); bezyl
N-acetyl-
-D-galactosaminide (BG), GlcNac Kit
from Oxford Glycosystems (Wakefield, MA); Coomassie reagent from Pierce
(Rockford, IL); alkaline phosphatase, peroxidase-conjugated goat
anti-rabbit IgG antibody from Roche Diagnostic (Indianapolis, IN);
bisbenzimide (Hoechst no. 33258), catalytic protein kinase A
(cPKA), forskolin (FSK), fraction V bovine albumin, genistein,
globulin-free bovine albumin, IBMX, NP-40, protease inhibitor cocktail
(PIC), staurosporine, and common reagents and salts from Sigma Chemical
(St. Louis, MO); antibiotic solutions and cell culture media from the
Tissue Culture Support Center, Washington University (St. Louis, MO); protein kinase A inhibitor (PKAI) from UBI (Lake Placid, NY); and Oct-1
(C-21) antibody from Zymed Laboratories (San Francisco, CA).
Preparation and culture of stably transfected INS-1 cells that
overexpress islet iPLA2.
A retroviral system (6, 23) was used to stably transfect
INS-1 cells (at ~passage 70) with iPLA2
cDNA and
achieve overexpression, as described (22). Freshly
collected retrovirus-containing medium was added to INS-1 cell
monolayers, and stably transfected cells expressing high levels of
iPLA2
were selected by culturing retrovirally infected
cells with medium containing G-418 (0.4 mg/ml). The expanded iPLA2
-overexpressing INS-1 cells were cultured, as
described (11), in RPMI 1640 medium, which contained 11 mM
glucose, 10% fetal calf serum, 10 mM HEPES buffer, 2 mM glutamine, 1 mM sodium pyruvate, 50 mM
-mercaptoethanol (BME), and 0.1% (wt/vol)
each of penicillin, fungizone, and streptomycin. RetroPack PT 67 cells (Clontech, Palo Alto, CA) were maintained in DMEM (4.5 mg/ml glucose) containing 10% FBS, 4 mM L-glutamine, 100 U/ml of
penicillin, and 100 µg/ml of streptomycin.
Preparation of INS-1 cell nuclear fraction.
iPLA2-overexpressing INS-1 cells, grown to confluence in
T75 flasks, were washed with phosphate-buffered saline (PBS) and detached by incubation (3 min, 37°C) with 0.05% trypsin-0.02% EDTA.
The cells were transferred to Tris-buffered saline (TBS) and pelleted
by centrifugation (1,500 g for 5 min). Nuclear extracts were
prepared, as described (42), by resuspending the cells in
400 µl of cold buffer A [10 mM HEPES, pH 7.9, 10 mM KCl,
0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT) and 10 µl
PIC/106 cells] by gentle trituration. The cells were
allowed to swell on ice for 15 min, and then 10 µl of freshly
prepared 1% NP-40 (final concentration 0.025%) were added. After
vigorous vortexing for 10 s, the cell homogenate (CH) was
centrifuged (16,000 g, 30 s, 4°C). The supernatant,
containing cytoplasm and RNA, was saved and designated nonnuclear (NN)
material. The nuclear pellet was resuspended in 50 µl of ice-cold
buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT, and PIC) and rocked vigorously (15 min, 4°C). The
nuclear homogenate (NH) was centrifuged (16,000 g, 5 min,
4°C), the nuclear extract (NE) supernatant was saved, and the
particulate (P) material was resuspended in buffer B (25 µl). Protein content in each fraction was determined by Bio-Rad assay
against the BSA standard.
Verification of purity of nuclear preparation.
To ensure that the nuclear fraction did not contain mitochondria or
plasma membrane fractions that might contribute to measured iPLA2 activity, cytochrome c oxidase
(mitochondrial marker enzyme) and 5'-nucleotidase (plasma membrane
marker enzyme) activity assays were performed, as described (33,
42). In addition, aliquots of the cellular fractions were
processed for immunoaffinity analyses, as we will describe, for the
nuclear marker Oct-1. Incubations with primary (1:1,000) and secondary
(1:40,000) antibodies were followed by visualization of
immunoreactive bands by ECL.
iPLA2 enzymatic activity assay.
Enzymatic Ca2+-independent PLA2
activity in
aliquots of cellular fractions (30 µg of protein) were assayed by
ethanolic injection (5 µl) of the substrate PLPC (5 µM) in assay
buffer (40 mM Tris, pH 7.5, and 5 mM EGTA, total volume 200 µl).
Assay mixtures were incubated (5 min, 37°C, with shaking), and the
assay reaction was terminated with butanol (100 µl) addition and
vigorous vortexing. The reaction mixture was centrifuged (2,000 g, 5 min), and products in the upper butanol layer were
analyzed by silica gel G thin-layer chromatography (TLC) in petroleum
ether-ethyl ether-acetic acid (80/20/1). The TLC plate region
containing free fatty acid was identified with iodine vapor and
scraped, and the released 14C fatty acid was quantitated by
liquid scintillation spectrometry. Specific iPLA2
activity was calculated from the dpm of released fatty acid and protein
content as described (14). To verify that the measured
activity reflected that of the iPLA2
(35), the abilities of ATP (10 mM) to stimulate and of BEL (10 µM), a
suicide inhibitor of iPLA2
, to inhibit activity were
examined. To test the effects of BEL on activity, the sample protein
was preincubated with BEL [2 min at room temperature (RT)] before addition of substrate.
Immunoaffinity analyses of INS-1 cell iPLA2
protein.
INS-1 cellular fractions were diluted in an equal volume of sample
buffer (1 M Tris · HCl, pH 6.8, 69 mM SDS, 10% glycerol, 0.01% bromophenyl blue, and 1% BME) and boiled. Aliquots containing 25-50 µg of protein were analyzed by SDS-PAGE (7.5%) and
transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membrane.
The electroblot was sequentially blocked (3 h at RT) with TBS-Tween 20 (TBS-T; 20 mM Tris · HCl, 137 mM NaCl, pH 7.6, and 0.05%
Tween-20) containing 5% milk protein, washed (TBS-T, 5 × 5 min),
and then incubated (1 h, RT) with purified polyclonal (p) antibodies
(0.0015 µg/µl in TBS-T containing 3% BSA) to iPLA2
generated by multiple antigen core technology (Research Genetics,
Huntsville, AL) against peptides in the iPLA2
-deduced
amino acid sequence. The iPLA2
peptides coupled to this
core for immunizing rabbits were
25KEVSLADYASSERVRE41 and
489RMKDEVFRGSRPY502. The membrane was again
washed in TBS-T (5 × 5 min) and incubated (1 h, RT) with
peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:40,000
in TBS-T containing 3% BSA). The immunoreactive protein bands were
visualized by ECL.
Visualization of iPLA2 protein by
immunofluorescence staining and confocal microscopy.
After removal of media, the attached cells were sequentially washed
with PBS (2 × 5 min), fixed in 4% paraformaldehyde (10 min),
washed (3 × 5 min), permeabilized in cold methanol (20 min,
20°C), washed (3 × 5 min), and blocked (in PBS containing 1% globulin-free BSA, 0.30% Triton X-100, and 3% normal goat serum). Next, piPLA2
primary antibodies (0.003 µg/µl) were
added, and the cells were incubated in a humidified chamber (O/N,
4°C). The cells were washed free of the primary antibodies (3 × 5 min) and incubated with fluorescent Cy3-conjugated AffiniPure goat
anti-rabbit IgG secondary antibody (1:400, 1 h). The cells were
then washed (PBS, 3 × 5 min) and covered with a drop of antifade
solution, and the slides were mounted with coverslips. Cell
iPLA2
immunofluorescence was visualized by confocal
microscopy at an excitation wavelength of 550 nm and an emission
wavelength of 570 nm.
Visualization of nuclear localization of iPLA2 by
dual-labeling fluorescence.
To examine whether the iPLA2
protein was accumulating in
the nuclear region, after incubation with Cy3, the cells were washed (PBS, 2 × 5 min) and further incubated (12 min, RT) with
bisbenzimide (1:20,000), a fluorescent DNA stain that
intercalates in A-T regions of DNA. The cells were washed (PBS, 3 × 5 min) and covered with a drop of antifade solution, and the slides
were mounted with coverslips. Rhodamine and DAPI filters attached to a
Nikon Eclipse TE 300 Inverted Scope were used to visualize Cy3
(iPLA2
, red) and bisbenzimide (nuclei, blue)
fluorescences, respectively, separately and in combination.
Effects of glucose, IBMX, and FSK stimulation on insulin
secretion and iPLA2 protein localization.
At confluence, iPLA2
-overexpressing cells were detached
from T75 flasks, and aliquots of cells (~2 · 104)
were seeded in multichambered glass slides and allowed to attach overnight. The culture medium was then removed, and the cells were
washed twice in Krebs-Ringer buffer (KRB), pH 7.3, containing (in mM):
115 NaCl, 24 NaHCO3, 5 KCl, 1 MgCl2, 25 HEPES,
1 glucose, and 0.10% BSA and were incubated in the same medium (1 h,
37°C, under an atmosphere of 95% air-5% CO2). The
medium was then removed and replaced with KRB medium containing glucose
(0-20 mM) without or with IBMX (100 µM) or FSK (2.5 µM). The
cells were further incubated (0-60 min, 37°C, under an
atmosphere of 95% air-5% CO2). At the end of the
incubation period, the medium was removed for measurement of insulin by
radioimmunoassay (65), and the cells were harvested for
iPLA2
enzymatic activity assays, as we have described.
In vitro phosphorylation of iPLA2.
Islet iPLA2
was overexpressed in and purified from sf9
insect cells by use of sequential (DEAE anion-exchange, ATP-affinity, and calmodulin-affinity) column chromatography, as previously described (49). Aliquots of the purified protein were
incubated (30 min, 37°C) with buffer (10 mM Tris, 10 mM MgAc, 4 mM
EGTA, pH 7.20), cPKA (10 U), and 10 µM [
-32P]ATP
(total volume 110 µl), without and with either PKAI (25 µM), the tyrosine phosphorylation inhibitor genistein (100 µM), or
staurosporine (100 nM), a nonspecific inhibitor of phosphorylation. After the assay period, the samples were immediately diluted in sample
buffer and boiled. Aliquots of the samples were then analyzed by
SDS-PAGE (7.5%), and the protein from gels was transferred onto
Immobilon-P PVDF membrane. The transferred 32P-labeled
proteins were visualized by autoradiography. Subsequently, the same
blot was processed for iPLA2
immunoaffinity analyses, as
we have described.
In vitro glycosylation of purified iPLA2.
To detect the presence of O-GlcNac modification of the
iPLA2
, protein transfer of radiolabeled galactose onto
the iPLA2
protein in the presence of
-D-galactosyltransferase was examined using reagents
supplied in the GalNac kit. Aliquots of purified iPLA2
or standard hen egg albumin were incubated (4°C, 3 h, total volume of 50 µl) with reaction buffer B, consisting of 5 mM MnCl2, UDP [3H]galactose (sp act 5-20
mCi/mmol), and
-D-galactosyltransferase (0.025 U/ml).
The reaction was terminated by the addition of sample buffer. Aliquots
of the reaction mixtures were analyzed by SDS-PAGE, and the gel was
fixed (30% MeOH/7% HAc, 15 min), soaked in Enhancer solution (30 min)
and then in 10% glycerol (30 min), dried (80°C, 1 h), and
cooled (10 min). Then followed visualization of labeled protein by autoradiography.
Effects of inhibition of PKA and O-glycosylation on insulin
secretion, iPLA2 protein localization, and enzymatic
activity.
To examine whether phosphorylation or glycosylation events contribute
to iPLA2
protein translocation in the cell, cellular permeable inhibitors of PKA (H-89) and of O-glycosylation
(BG) were utilized. INS-1 cells overexpressing iPLA2
were seeded in 24-well plates (for insulin secretion assays), flasks
(for enzymatic activity assays), or glass slides (for
immunofluorescence assays), and were pretreated with either H-89
(10-20 µM, 60 min) or BG (2-4 mM, 24 h), prepared in
DMSO. The inhibitors were also included during stimulation of the cells
with glucose (2 mM) in the absence and presence of FSK (2.5 µM).
Control cells were incubated with the vehicle DMSO alone. After the
incubation period, the medium was collected for measurement of insulin
content, and the cells were processed either for enzymatic activity
assays or immunofluorescence analyses.
Statistical analyses. The data were converted to mean ± SE values where appropriate, and the Student's t-test was applied to determine significant differences (at P < 0.05) between two groups.
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RESULTS |
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Enzymatic activity and protein expression of iPLA2
in transfected INS-1 cells.
To verify expression of active iPLA2
protein in INS-1
cells transfected with iPLA2
cDNA, homogenates were
prepared from INS-1 cells transfected with either an empty retroviral
(V) construct or with an iPLA2
cDNA (I)-containing
construct. Aliquots of protein from these fractions were used to
determine iPLA2
activity, by radiochemical enzymatic
assay, and iPLA2
protein expression, by immunoaffinity
analyses. As expected, homogenates prepared from INS-1 cells
transfected with iPLA2
cDNA expressed higher activity
(nearly 10-fold) than cells transfected with the V construct (Fig.
1A). The increased expression
of activity was reflected by the higher expression of
iPLA2
protein in the INS-1 cells transfected with
iPLA2
cDNA relative to V cells (Fig. 1B).
These results confirm that transfection of INS-1 cells with
iPLA2
cDNA results in higher expression of catalytically
active iPLA2
.
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Distribution of iPLA2 in overexpressing INS-1 cells.
Immunoaffinity, activity, and immunofluorescence analyses were next
used to examine in greater detail the distribution of iPLA2
within the INS-1 cells (Fig.
2). Cellular fractions prepared from
iPLA2
-overexpressing INS-1 cells, as described in
METHODS, were used in these analyses. As illustrated in
Fig. 2A, left, abundant immunoreactive iPLA2
protein was evident in the CH, NN (containing cytosol), and NE
fractions, with low iPLA2
protein content evident in the
P fraction. The specificity of the antibody affinity for
iPLA2
was then examined after incubations with secondary antibody alone (Fig. 2A, lane 1), preimmune IgG
(lane 2), or piPLA2
antibodies plus antigenic
peptide sequences of iPLA2
against which the antibodies
were generated (lane 3). Under all three conditions, no
signal for the iPLA2
protein was detected. These findings confirm that the piPLA2
antibodies generated by
Research Genetics strategy are specific for the iPLA2
protein. Figure 2A, right, reflects iPLA2
enzymatic activity in the corresponding INS-1 cell fractions and
illustrates an abundance of iPLA2
catalytic activity in
the nuclear fraction.
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Immunofluorescence analyses of iPLA2 distribution in
INS-1 cells after stimulation with glucose and IBMX.
To examine whether the iPLA2
protein undergoes
translocation under conditions that promote insulin secretion, parental
INS-1 cells and INS-1 cells transfected with iPLA2
cDNA
were stimulated with glucose and IBMX, either alone or in combination.
The cells were then sequentially fixed, permeabilized, and incubated
with piPLA2
antibodies followed by Cy3 fluorescent
secondary antibody. Localization of iPLA2
fluorescence
was then visualized by confocal microscopy. Fluorescence recordings for
iPLA2
in parental cells after a 60-min stimulation
period with glucose in the absence and presence of IBMX are presented
in Fig. 3. In the presence of no added
glucose or IBMX, iPLA2
fluorescence is diffuse
(A). At 2 mM glucose, the iPLA2
fluorescence
appears as a ring (B) in the nuclear region. Higher glucose
concentrations result in more prominent fluorescence, which is
distributed throughout the cytoplasm and nuclear regions (C
and D). Addition of IBMX by itself results in distinct
iPLA2
fluorescent ring formation (E), which becomes more diffuse with increasing concentrations of glucose (F-H).
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Visualization of nuclear accumulation of iPLA2 by
dual-signal immunofluorescence.
To verify that the stimulated iPLA2
signal was indeed
accumulating in the nuclear region, a dual-labeling fluorescence
protocol was used. INS-1 cells overexpressing iPLA2
were
stimulated with either glucose (2 mM) or IBMX (100 µM) alone and
subsequently processed for dual-fluorescence analyses. The
iPLA2
protein is visualized with Cy3 red fluorescence
and the nuclear region with bisbenzimide blue fluorescence.
An overlap of the two fluorescences would yield a pinkish hue and
reflect the appearance of the iPLA2
protein in the
nuclear region. This is illustrated in Fig.
5. In A, only the
iPLA2
fluorescence with 2 mM glucose is visualized; in
B, only the nuclear region in the corresponding cells is
visualized. When both fluorescences are simultaneously monitored
(C), a clear separation of the red iPLA2
from
the blue nuclear fluorescence is evident. Figure 5D depicts
the higher-intensity iPLA2
fluorescence in the presence
of IBMX alone, and Fig. 5E reveals the nuclear region in the
corresponding cells. When both fluorescences are simultaneously
monitored in the IBMX-stimulated cells (F), the nuclear blue
region is replaced by a pinkish coloring from the overlap of
iPLA2
and the nuclear region, suggesting that, in the
presence of IBMX, the iPLA2
protein accumulates in the
nuclear region.
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Verification of purity of nuclear preparation.
To determine whether the accumulation of iPLA2 protein
in the nuclear region was accompanied by increases in nuclear
iPLA2
activity, nuclear fractions were prepared from
iPLA2
-overexpressing INS-1 cells according to the
procedure of Schreiber et al. (41). Because we have
previously observed the presence of iPLA2
activity in
mitochondrial and plasma membrane compartments of
-cells
(14), the possibility that the nuclear preparation is
contaminated with these cellular components was first assessed. As
illustrated in Fig. 6,
mitochondria-associated cytochrome c oxidase (A)
and plasma membrane-associated 5'-nucleotidase (B)
activities are enriched in the NN fraction relative to the nuclear
fraction. Furthermore, immunoreactive nuclear transcription factor
Oct-1 is evident in the nuclear fraction (C) but not in the
NN fraction. These findings verify that the NE fraction is not
contaminated with mitochondria or plasma membrane and contains nuclei.
To verify that the Ca2+-independent PLA2
activity expressed in NN and NE fractions is manifested by the
iPLA2
protein, the ability of ATP to stimulate and of
BEL, a suicide inhibitor of iPLA2
(35), to
inhibit activity was determined. The
-cell iPLA2
enzymatic activity has previously been shown to be uniquely sensitive
to ATP and BEL (35-37). As demonstrated in Fig.
6D, the enzymatic activity expressed in both fractions is
found to be stimulated by ATP and completely inhibited by BEL; arrows
indicate absence of measurable activity. These findings therefore
confirm the presence of iPLA2
protein in the INS-1 cell
fractions of interest.
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Stimulated accumulation of nuclear iPLA2 enzymatic
activity.
To examine more directly the influence of cAMP-elevating agents on the
distribution of iPLA2
in overexpressing cells, the adenylate cyclase stimulator FSK was utilized in subsequent studies. Hence, iPLA2
activity was determined in the NN and NE
fractions after treatment of iPLA2
-overexpressing INS-1
cells with glucose (2 mM) in the absence and presence of FSK. As
illustrated in Fig. 7A,
NN-associated specific iPLA2
activity is relatively
unchanged under 2 G ± FSK conditions. This is confirmed in
immunoaffinity analyses, which reveals no significant change in the
iPLA2
content in this fraction (data not presented). In
contrast, nuclear-associated iPLA2
activity increases
only modestly (Fig. 7B) with 2 G, but significantly (nearly
2-fold) with the addition of FSK. Such an increase in activity is
reflected by increased accumulation of the iPLA2
protein
(Fig. 7C) in the NE fraction after stimulation with FSK.
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Potential phosphorylation and glycosylation
of iPLA2.
Previous reports suggest that phosphorylation stimulates nuclear
association of catalytic (c) PLA2 (13, 40),
and that cytosolic and nuclear proteins undergo stimulated
glycosylation (7, 15). In view of this, it was of interest
to examine the potential involvement of these two processes in nuclear
accumulation of iPLA2
in INS-1 cells. Elevations in
cAMP, as with FSK, induce cAMP-dependent PKA-induced phosphorylation of
serine and threonine residues of proteins (8). As such,
the ability of cPKA to phosphorylate iPLA2
was examined.
Treatment of purified iPLA2
with cPKA promotes incorporation of 32P into the protein (Fig.
8A, top, lane
1). Such PKA-induced phosphorylation of iPLA2
is
completely blocked by a specific peptide inhibitor of PKA (PKAI,
lane 2) and by the nonspecific inhibitor of phosphorylation, staurosporine (lane 5) but not by genestein (lane
3), which inhibits tyrosine phosphorylation. As shown
(bottom), immunoaffinity analysis of the same blot verifies
that all lanes are loaded with similar iPLA2
protein
amounts. As illustrated in Fig. 8B, incubation of purified
iPLA2
with galactosyltransferase results in
glycosylation of the protein. These findings raise the possibility that
-cell iPLA2
is a candidate for phosphorylation and
glycosylation.
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Involvement of protein phosphorylation and glycosylation during
insulin secretion.
To examine whether protein phosphorylation or glycosylation has a role
during stimulation of insulin secretion from
iPLA2-overexpressing INS-1 cells, the effects of H-89
and BG, cell-permeable specific inhibitors of PKA (50) and
O-glycosylation (20), respectively, were
examined. As shown in Fig. 9, insulin
secretion from the overexpressing INS-1 cells is modestly elevated in
the presence of 5 mM glucose alone, but robustly amplified with
additions of cAMP-elevating agents IBMX and FSK. Both H-89 and BG
significantly attenuate the IBMX- and FSK-induced responses, suggesting
that phosphorylation and glycosylation events participate in insulin- secretory responses amplified by cAMP-elevating agents.
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Effects of inhibitors of phosphorylation and glycosylation on
iPLA2 localization.
Immunofluorescence analyses were performed next to examine whether
inhibition of protein phosphorylation or glycosylation affects
stimulated nuclear accumulation of iPLA2
. As illustrated in Fig. 10, relative to the 0 G
condition (A), presence of 2 G alone promotes appearance of
a modest ring-like accumulation of iPLA2
in the nuclear
region (B). Neither H-89 (C) nor BG
(D) appears to affect such accumulation. FSK alone
(E), similar to IBMX (Fig. 4), increases nuclear
accumulation of the iPLA2
protein, and addition of 2 G
further promotes this association into ring-like formation
(F). After treatment with H-89 (G) or BG
(H), FSK-amplified increases in nuclear immunofluorescence
of iPLA2
are markedly reduced. When iPLA2
enzymatic activity was determined in the corresponding groups,
FSK-stimulated increase in nuclear iPLA2
activity was
found to return to basal levels (Fig.
11). Collectively, these findings
suggest that phosphorylation and glycosylation events participate in
stimulated nuclear accumulation of iPLA2
in INS-1
-cells.
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DISCUSSION |
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Potential functions proposed for the -isoform of
calcium-independent Group VIA PLA2
(iPLA2
) include generation of substrate for
eicosanoid synthesis (28), membrane degradation
during apoptosis (2), regulation of
phosphatidylcholine composition and content (4, 5),
and involvement in signaling events (19, 46).
Studies in murine P388D1 macrophage-like cells have led to the
suggestion that iPLA2 participates in phospholipid
remodeling and therefore serves a housekeeping role (4).
In that study, they observed that inhibition of iPLA2
activity with either BEL or with antisense oligonucleotide treatments
led to suppressed incorporation of arachidonic acid into the
phosphatidylcholine pool and also reduced the generation of
lysophosphatidylcholine (4). Subsequent studies in our
laboratory, however, indicate that suppression of iPLA2
inhibits insulin secretion without affecting arachidonic acid
incorporation into the
-cell phosphatidylcholine pool
(35). Furthermore, when the proposed role of
iPLA2
in phospholipid remodeling was examined in INS-1
insulinoma cells overexpressing iPLA2
, the rate or
extent of arachidonic acid incorporation into INS-1 cell phospholipids
was not found to be altered, although the insulin-secretory response
was markedly greater in the iPLA2
-overexpressing INS-1
cells (22). These findings strongly suggest that the
iPLA2
enzyme manifests a signaling, rather than a
housekeeping, role in
-cells.
The iPLA2-overexpressing INS-1 cells studied in the
present study were found to be more sensitive to cAMP-elevating agents than parental INS-1 cells. Increasing concentrations of glucose produced only modest increases in insulin secretion, but IBMX (a
phosphodiesterase inhibitor) and FSK (an adenyl cyclase activator) promoted robust increases in the insulin secretory response. Attenuated responses to glucose alone have previously been recognized in native
-cells that are stimulated after separation from the non-
-cell population of pancreatic islets (29). Glucose stimulation
of isolated islet
-cells promotes only minor increases in insulin secretion, but costimulation with cAMP-elevating agents or
reintroduction of non-
-cells restores the insulin-secretory capacity
(31). The ability of cAMP-elevating agents to amplify
glucose-stimulated insulin secretion has also been noted in insulinoma
cell lines (32). These observations highlight the
requirement for maintaining a critical level of cAMP for
glucose-stimulated insulin secretion.
Overexpression of iPLA2 in INS-1 cells promotes
accumulation of the enzyme in the nuclear region, even under basal
conditions. Additions of increasing concentrations of glucose do not
influence this accumulation significantly, but cAMP-elevating agents
dramatically enhance accumulation of iPLA2
in the
nuclear region, as reflected by immunofluorescence, immunoaffinity, and
enzymatic activity analyses. This effect is time dependent and very
closely correlates with the secretory response seen in the
iPLA2
-overexpressing cells. Such a stimulated
translocation of iPLA2
provides further evidence to
support its potential role in signaling during secretory events.
Nuclear association of iPLA2 induced by cAMP-elevating
agents in INS-1 cells is of interest, because glucose promotes
-cell insulin secretion and proliferation, and glucose-induced INS-1 cell
mitogenesis is cAMP dependent (18). As membranes of the nucleus and endoplasmic reticulum (ER) are contiguous (17,
40), perinuclear accumulation of iPLA2
is
consistent with association with a subcellular compartment that is
likely to include ER (17). The
-cell ER is known to
contain an abundance of arachidonate-containing plasmenylethanolamine
molecular species (33), and products of PLA2
action induce Ca2+ release from
-cell ER
(48), which is thought to participate in induction of
insulin secretion (10). cAMP-mediated increases in
cytosolic [Ca2+], via Ca2+ entry and
mobilization of intracellular Ca2+ stores (11, 17,
51), and sensitization of the exocytotic apparatus to
Ca2+ (1) are among the mechanisms by which
cAMP augments the insulin- secretory responses. In view of earlier
observations suggesting involvement of arachidonic acid, in particular
of arachidonic acid hydrolyzed from plasmenylethanolamine molecular
species (33, 34) and of its metabolites in the
-cell
secretory process (45), the nuclear association of
iPLA2
after stimulation with cAMP-elevating agents might
be consistent with a process that results in the generation of
arachidonic acid in close proximity to enzymes, which catalyze
eicosanoid generation, localized in the nuclear envelope and ER
(27, 39).
The nuclear association of cPLA2 after cellular stimulation
with the calcium ionophore A-23187 has been reported to be associated with increased phosphorylation of the cPLA2 (13,
40). Although nuclear accumulation of iPLA2 has
been reported during myocardial ischemia (47), it
is not known whether nuclear accumulation is associated with
phosphorylation of the iPLA2
. Because stimulation with
IBMX or FSK increases cAMP content in INS-1 cells (22), it
might be speculated that, as a consequence, a cAMP-dependent PKA, which
phosphorylates serine and threonine residues, is activated and that the
iPLA2
serves as a candidate for PKA-catalyzed
phosphorylation. The likelihood that the iPLA2
becomes
associated with the nuclear region is enhanced by the recognition of
a bipartite nuclear localization sequence
(511KREFGEHTKMTDVKKPK527)
in the deduced amino acid sequence of iPLA2
(24) that is similar to the sequence in other nuclear
proteins (10, 38).
To examine the possibility that iPLA2 can undergo
PKA-stimulated phosphorylation, islet iPLA2
overexpressed in sf9 cells and purified was incubated with cPKA. Under
the conditions studied, PKA promotes phosphorylation of
iPLA2
in a protein concentration-dependent manner (data
not shown). Such phosphorylation is completely blocked by a
specific peptide inhibitor of PKA and by the general inhibitor of
phosphorylation staurosporine, but not by genistein, which inhibits
tyrosine phosphorylation. These findings raise the possibility that
iPLA2
is a candidate for PKA-induced phosphorylation. In view of this, effects of inhibiting cellular PKA activity with H-89 (a
cell-permeable inhibitor) on iPLA2
distribution were examined. The data obtained in the presence of H-89 reveal that FSK-stimulated accumulation of iPLA2
immunofluorescence
in the nuclear region is dramatically attenuated and that this is
accompanied by reductions in FSK-stimulated nuclear accumulation of
iPLA2
enzymatic activity and by inhibition of
cAMP-elevating agent-stimulated insulin secretion.
Another potential modification of the iPLA2 protein that
might affect its localization is glycosylation, a modification that has
been reported with several cytosolic and nuclear proteins (7,
15). Although it is not clear what effect glycosylation of the
iPLA2
protein might have on its function or
localization, it might be noted that the iPLA2
contains
a consensus sequence site for O-GlcNacylation.
Interestingly, iPLA2
also contains ankyrin-like repeat
sequences (25) that bind tubulin and integral membrane
proteins (30), and O-GlcNacylation is involved
in neurofilament assembly (16). A number of agents that
interfere with microtubule assembly or disassembly inhibit insulin
secretion (21). In view of these observations, it might be
speculated that glycosylation of the iPLA2
promotes its
binding to the neurofilament processes through the ankyrin-like repeats
and facilitates its trafficking into the nuclear region. Such a
possibility was examined using purified iPLA2
and intact
cells. Incubation of purified iPLA2
with
galactosyltransferase resulted in an increase in the incorporation of
labeled galactose, suggesting that the protein was a candidate for
O-GlcNacylation. After treatment of
iPLA2
-overexpressing cells with BG, a cell-permeable
competitive inhibitor of galactosaminyltransferase and
O-glycosylation, decreases in FSK-stimulated nuclear
accumulation of iPLA2
fluorescence are observed. As with
H-89, FSK-stimulated nuclear accumulation of iPLA2
activity and insulin secretion are also significantly attenuated by BG.
Collectively, the data obtained with H-89 and BG raise the possibility
that phosphorylation and glycosylation of the iPLA2
protein itself, or of associated proteins involved in the secretory
pathway, might participate in promoting translocation of
iPLA2
to the nuclear region.
In summary, in the present study we report that the -cell
iPLA2
becomes associated with the nuclear region under
conditions that promote increases in insulin secretion.
Posttranslational modifications such as phosphorylation and
glycosylation may potentially participate in promoting its association
with the nuclear region and facilitate its signaling role in
-cells.
Detailed analyses are still required to identify the sites in the
iPLA2
protein that might be modified during stimulatory
conditions, and such studies are currently underway.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Karen Green, Dr. William Cruz, and Dr. Burton Wice for their advice on immunofluorescence assays; Sam Smith and Dr. Kevin Yarasheski for their assistance with the cytochrome c oxidase assay; and Alan Bohrer and Dr. Mary Wohltmann for their expert technical assistance.
![]() |
FOOTNOTES |
---|
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34388, a Career Development Award (to Z. Ma) from the Juvenile Diabetes Foundation (no. 2-1999-55), and a Career Development Award (to S. Ramanadham) from the American Diabetes Association.
Address for reprint requests and other correspondence: S. Ramanadham, Washington Univ. School of Medicine, Dept. of Medicine, Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: sramanad{at}im.wustl.edu).
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.
10.1152/ajpendo.00165.2001
Received 10 April 2001; accepted in final form 16 November 2001.
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