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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3833-3840
Novel Evidence of Expression and Activity of Ecto-Phospholipase C
 1 in Human T Lymphocytes
By
Sebastiano Miscia,
Angela Di Baldassarre,
Amelia Cataldi,
Rosa Alba Rana,
Valerio Di Valerio, and
Giuseppe Sabatino
From the Istituto di Morfologia Umana Normale and Cattedra di
Neonatologia, Università G. D'Annunzio, Chieti, Italy.
 |
ABSTRACT |
Although much is known about the intracellular phospholipase C (PLC)
specific for inositol phospholipids, few data are available about the
presence of a less common PLC at the external side of the membrane
bilayer of some cell types. This ectoenzyme seems to play particular
roles in cellular function by hydrolyzing inositol lipids located on
the outer leaflet of the plasma membrane. Here, we provide the first
evidence that peripheral T lymphocytes express a discrete level of a
PLC 1 at the outer leaflet of the plasma membrane. Flow cytometry
showed that the PLC1-positive (PLC1+) cells
(~37%) were CD8+ and CD45RA+.
Biochemical evidence indicated that (1) this ectoenzyme displays a mass
similar to the cytoplasmic form, (2) it is phosphorylated on tyrosine
residues, and (3) its activity is Ca2+-dependent. In
addition, this enzyme appeared to be correlated with the proliferative
state of the cell, since stimulation with phytohemagglutinin (PHA)
downregulated both its expression and activity, which were restored by
treatment with an antiproliferative agent like natural interferon beta.
Moreover, the different kinetics of formation of its hydrolytic
products, inositol 1 phosphate and inositol 1:2 cyclic phosphate
(Ins(1)P and Ins(1:2 cycl)P), formed upon incubation of the lymphocytes
with [3H]-lyso-phosphatidylinositol (PI), allow the hypothesis of a
selective involvement of the two inositol phosphates in the mechanisms
regulating the metabolism of particular T-lymphocyte subsets.
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INTRODUCTION |
IT IS WELL ESTABLISHED that
phosphatidylinositol (PI)-specific phospholipase C (PLC) plays a
pivotal role in signal transduction in a wide range of cellular
systems.1-5 PLC-dependent phosphoinositide hydrolysis
occurs both at the plasma membrane and in the nuclear compartment,6 and most of the relevant research has focused on the role of intracellular PLC isoform activity. Only limited data
are available about the less common expression of PLC at the external
surface of some cell types.7-11 This surface activity may
be involved in the regulation of cell growth.12-14 Here, we report the first evidence of substantial levels in human T lymphocytes of cell surface PLC (ecto-PLC) isoform 1. We also report that its
expression varies during the modulation of cell proliferation. This
analysis was accomplished by means of confocal microscopy and flow
cytometry. In a parallel study, we tested the activity of this external
enzyme using inositol 2 3H-monoacyl-PI ([3H]-lyso-PI) as
a substrate and analyzing the formation of the specific hydrolytic
products.
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MATERIALS AND METHODS |
Cell culture and interferon treatment.
Lymphocytes were obtained from normal blood donors by
Ficoll-Hypaque separation (Sigma Chemical Co, St Louis, MO). To test normal resting T lymphocytes, samples were previously depleted from
adherent cells by incubating the cell suspension in tissue culture
dishes overnight at 37°C in a controlled atmosphere. For polyclonal
activation, cells were cultured in HEPES-buffered RPMI 1640, 10% fetal
calf serum, glutamine, and antibiotics in the presence of 20 µL/mL
phytohemagglutinin (PHA) at 37°C in 5% CO2 and high
humidity. For the experiments, human interferon beta (Serono
Co, Geneva, Switzerland; 1,000 IU/mL) was administered for
different times after 24 hours of PHA stimulation. Viability of the
cells was determined by the Trypan blue exclusion test, which indicated
that 95% of the cells were still viable after 4 days of culture.
Immunocytochemical and confocal analysis.
Lymphocytes were washed in phosphate-buffered saline (PBS) and
incubated with anti-PLC1,  1, or  1 MoAb (Upstate Biotechnology Inc, Lake Placid, NY; 1:300) and then with
fluorescein-conjugated goat Fab anti-mouse IgG (Sigma;
1:64) for 60 minutes. The samples were always kept in an ice bath
during the incubation period to avoid the capping and/or
internalization of Ag-Ab complex. All antibody solutions were diluted
in PBS and 4% goat and 4% human Ig to avoid cross-reactivity or
unspecific binding. The reactions performed using either anti-CD2 MoAb
(anti-leu-5b, 1:300 as a cell surface marker; Becton
Dickinson, Meylan, France) or anti-STAT 91/84 protein
(Affinity, Mamhead, Exeter, UK; 5 µg/mL as intracellular marker to
check integrity of the membrane) instead of the primary antibody or
only with the secondary antibody represented the controls for the
experimental procedures. In addition, to better characterize the
identity of the ectoenzyme, a different anti-PLC1 (Santa Cruz, Santa
Cruz, CA; 1:100) was tested. After washing in PBS, a drop
of each sample was layered on a slide, mounted, and observed with a
Leica TCS 4D equipped with an argon ion laser (Leica,
Heidelberg, Germany), attached to Leitz DMRB fitted with
a 100×/1.3 NA oil immersion objective (Leitz, Heidelberg, Germany).
For the image acquisition, fluorescein isothiocyanate (FITC) was
excited with the blue (488 nm) line of the argon ion laser. Images were
stored on a computer with a scanning mode format of 512 × 512 pixels.
Multicolor immunofluorescence staining and flow cytometric analysis.
Peripheral blood mononuclear cells (PBMCs) were isolated from
heparinized venous blood by Ficoll-Hypaque density gradient centrifugation. Two-color immunofluorescence was used to examine PLC1 expression by CD4+, CD8+,
CD45RA+, and CD45RO+ cells. PBMCs were first
treated with anti-PLC1 (UBI) or, as a control, with mouse IgG
(Coulter, Miami, FL) and counterstained with F(ab )
fragments of FITC-conjugated anti-mouse IgG (Sigma). Next, the cells
were stained for determination of T-cell subpopulations with
phycoerythrin (PE)-conjugated anti-CD4, -CD8, -CD45RA, and -CD45RO
MoAbs (Sigma) or isotype-matched mouse IgG. Cells were analyzed on a
fluorescent-activated cell scan (FACScan) using Lysis II software
(Becton Dickinson). The region for the lymphoid population was then
selected. Ten thousand events were accumulated and analyzed for
fluorescence.
Preparation of cell extracts.
Resting T lymphocytes were washed in PBS, homogenized in a
homogenization buffer (20 mmol/L Tris, pH 7.4, 100 mmol/L NaCl, 5 mmol/L MgCl2, 0.2 mmol/L Na3VO4, 10 µg/mL aprotinin, and 10 µg/mL leupeptin), and kept for 30 minutes
on ice. The lysates were centrifuged at 8,000 g for 15 minutes
at 4°C. The supernatants were recovered as the cytosolic fraction.
Homogenization buffer containing 50 mmol/L Triton X-100 was added to
the particulate fraction to solubilize membrane proteins. After
incubation at 22°C for 20 minutes, samples were centrifuged at
400,000 g for 20 minutes at 4°C, and the supernatants were
collected as the solubilized membrane fraction.
Immunoprecipitation of PLC1.
Lysates from membrane and cytosolic fractions normalized at 400 µg
protein were incubated at 4°C for 60 minutes with 10 µg anti-PLC1 MoAb (UBI) previously coupled to goat anti-mouse IgG magnetic beads. Immunocomplexes were collected by a magnet and washed
several times with ristocetin-induced platelet
agglutination buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate,
and 0.1% sodium dodecyl sulfate [SDS]) in the presence of protease
inhibitors. Equal amounts of immunocomplexes were subjected to
immunoblot analysis.
Immunoblot analysis.
Cytosolic and membrane extracts or equal amounts of PLC1
immunocomplexes from the two fractions were diluted in SDS sample buffer, electrophoresed in 8% SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto nitrocellulose membrane, and incubated for 1 hour at room temperature with anti-PLC1 antibody (UBI or Santa Cruz;
1 µg/mL) or anti-phosphotyrosine (PY-20) MoAb (Affinity; 1:1,000) in
wash buffer (10 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20),
and 5% nonfat milk. Immunoreactive bands were detected by a
chemiluminescence system (ECL; Amersham Corp, Buckinghamshire, UK) using peroxidase-conjugated secondary antibodies
diluted in wash buffer with 5% nonfat milk.
When required, blots were stripped of bound antibodies by incubating
the membranes in wash buffer containing 2% SDS at 80°C for 40 minutes. After washing, membranes were reprobed with the secondary
antibody to confirm removal of the primary antibody. Blots were then
incubated with anti-PLC1, and the antibody binding was detected by
the ECL system as previously described.
Internal controls obtained by incubating the membranes only with
secondary antibodies always yielded negative results.
Preparation of radiolabeled lyso-PI.
Tritium-labeled lyso-PI was prepared as described by Volwerk et
al.13 PI conversion to lyso-PI and complete removal of
fatty acids were verified by thin-layer chromatography
(chloroform:methanol:water 65:25:4). [3H]-lyso-PI was dried,
dissolved in water at 1.125 mmol/L, and stored frozen at  20°C
until use. The specific activity (5.50 Ci/mol) was verified by liquid
scintillation counting.
Kinetic analysis of [3H]-lyso-PI uptake and ecto-PLC activity.
Ecto-PLC activity was determined as described by Volwerk et
al.13 An equal number of lymphocytes
(1 × 106) in the different experimental conditions
(ie, unstimulated, PHA-stimulated, and PHA-stimulated plus 30 minutes,
90 minutes, 6 hours, and 24 hours of interferon beta, respectively)
were incubated at 4°C up to 30 minutes with 11.25 µmol/L
[3H]-lyso-PI. At the indicated times, aliquots from the samples were
centrifuged and the amount of water-soluble radiolabeled products
formed was measured by extracting a portion of the incubation medium
with chloroform:methanol:concentrated HCl (66:33:1). Since lyso-PI is
highly water-soluble, the aqueous phase was extracted again with
chloroform so that removal of this lipid was more complete. The aqueous
phase was then used for liquid scintillation counting. As controls,
samples containing EGTA (0.5 mmol/L) were always tested in parallel.
Direct scintillation counting of the incubation medium of these
EGTA-containing samples yielded the measure of spontaneous uptake of
lyso-PI by the cells.
Identification of inositol phosphate products.
Identification of inositol phosphates produced from hydrolysis of
[3H]-lyso-PI by the ecto-PLC of T lymphocytes was performed by
loading the aqueous phase onto a small (1 mL) Dowex
AG1-X8 anion-exchange resin (formate form) column
(Bio-Rad, Hercules, CA) as described by Berridge et al.15
The columns were previously calibrated with standard samples consisting
of [3H]-lyso-PI, sn-glycero-3-phospho-D-1 myo-inositol (Sigma), DL-myo-inositol-1
monophosphate (Sigma), and DL-myo-inositol 1,2 cyclic monophosphate (Sigma). Eluted fractions were collected and
processed for scintillation counting.
| |
RESULTS AND DISCUSSION |
The presence of PLC1 at the external side of T lymphocytes was
revealed by confocal microscopy on unfixed cells as a bright ring-shaped fluorescence (Fig 1A). The
reaction accomplished with the anti-PLC1 and -PLC1 MoAbs always
yielded negative results. Flow cytometric analysis showed that
PLC1+ cells represented 36.4% ± 4.8% of peripheral
lymphocytes (Fig 2). To assess whether the
enzyme expression correlated with given functional phenotypes, we then
analyzed PLC1 expression in specific subtypes of peripheral T
lymphocytes (CD4+, CD8+, CD45RA+,
and CD45RO+ cells). Flow cytometry was performed as
two-color analysis, and the results are expressed as the
percentage of total lymphocytes positive for a given phenotype.
PLC1+ cells were almost exclusively CD8+
cells, whereas the percentage of circulating
PLC1+CD4+ cells was found to be low (Fig
3A). To investigate whether expression of the enzyme was correlated
with "naive" or "memory" functional phenotypes, we extended
the analysis to CD45RA+ and CD45RO+
populations. The results indicated that PBMCs showed a significant percentage of PLC1+CD45RA+ cells, whereas
the ectoenzyme was expressed only on a small percentage of circulating
CD45RO+ cells (Fig 3B).
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| Fig 1.
Confocal analysis of PLC1 expression at the external
leaflet of the plasma membrane in human T lymphocytes. (A) Unstimulated cell (increments of 0.85 µm in z-axis; cell size, 8 µm); (B)
PHA-stimulated cell (increments of 0.9 µm in z-axis; cell size, 17 µm); (C) PHA-stimulated cell incubated for 90 minutes with interferon
beta (increments of 0.75 µm in z-axis; cell size, 14 µm). (D)
PHA-stimulated cell incubated for 24 hours with interferon beta
(increments of 0.54 µm in z-axis; cell size, 10 µm); (E) cell
labeled with anti-Leu-5b MoAb (CD 2), used as an internal control
(increments of 0.56 µm in z-axis; cell size, 11 µm). Note that the
membrane labeling evident in resting lymphocytes became weaker in
PHA-stimulated cells, and again was strongly detectable after addition
of interferon. As internal controls, cells were incubated with
anti-Leu-5b MoAb, a T-lymphocyte surface marker, which strongly labeled
the membranes (Fig 2E), while the reactions performed in the absence of
the primary antibody yielded consistently negative results. Results obtained by incubating lymphocytes with anti-STAT 91/84 protein as an
intracellular marker (to check membrane integrity) and with anti-PLC
and isoforms did not disclose any FITC membrane labeling.
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| Fig 2.
FACScan histogram of ecto-PLC1+
lymphocytes. The percentage of ecto-PLC1+ lymphocytes
was obtained by calculating the mean ± SD of 10 experiments.
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| Fig 3.
Expression of PLC1 at the external leaflet of the
plasma membrane in T-cell subsets. PBMCs were double-stained with
anti-PLC1, evidenced by a FITC-conjugated secondary antibody, and
with either PE-labeled anti-CD4, anti-CD8, anti-CD45RA, or anti-CD45RO
antibody. (A) Results showed that the enzyme is expressed on the
membrane of CD8+ cells (29.2% ± 5.1%), while the
percentage of circulating PLC1+CD4+
lymphocytes is very low (1.1% ± 0.2%, P < .05). (B)
When expression of the enzyme on naive and memory cells was considered,
PBMCs displayed a significant percentage of
PLC1+CD45RA+ (27.2% ± 1.7%),
whereas the phenotype PLC1+CD45RO+
was slightly detectable (4.2% ± 0.6%, P < .005).
Significance of the results was determined by Student's t
test. n = 10 samples.
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These results might suggest a regulation of the ectoenzyme along the
differentiation pathway from "naive" to "memory"
CD8+ cells, but this hypothesis requires accurate
investigation. To better characterize the apparently novel PLC1, we
performed an anti-PLC1 immunoblot analysis of the membrane and
cytosolic fractions from peripheral T lymphocytes. The anti-PLC1
MoAb showed a single band with a molecular weight that was apparently
the same in both extracts, thus indicating that the external PLC1
shares the same mass as the cytosolic one (Fig
4A). Since the immunocytochemical and
immunoblot detections were acquired using a mixed monoclonal preparation (anti-PLC1; UBI), to confirm the identity of the enzyme,
we repeated the reactions using a different anti-PLC1 antibody
(Santa Cruz). The latter, according to the manufacturer, is directed
against the epitope corresponding to amino acids 1249 to 1261 within
the carboxyl-terminal domain of PLC1. The results showed no
difference with respect to previous experiments (not shown), further
confirming the enzyme identity. In addition, these experiments
indicated that the enzyme exposes the above-reported epitope from the
external leaflet of the membrane, since it was clearly recognized by
the immunocytochemical reaction.
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| Fig 4.
(A) Immunoblot analysis of PLC1 in membranes and
cytosolic fractions from resting T lymphocytes. Proteins from membrane
and cytosolic fractions were separated by SDS-PAGE, blotted onto
nitrocellulose, and reacted with anti-PLC1 antibody. The ECL system
revealed for each fraction one band apparently at the same molecular
weight. Control is represented by a cell lysate from a
PLC1+ line (Jurkat); 145 corresponds to molecular
weight (in kilodaltons) assessed on the basis of standard comigration.
(B) Tyrosine phosphorylation level of PLC1 immunoprecipitated from
membranes and cytosolic fractions. PLC1 immunoprecipitated from
membrane and cytosolic fractions was probed with anti-phosphotyrosine
antibody (PY-20) and revealed by the ECL system. Membrane PLC1 was
consistently phosphorylated, while no or weak reactions were detected
in the cytosolic enzyme. After stripping anti-PY-20, the same blot was reprobed with anti-PLC1 antibody to show that the levels of the protein loaded on the gel were comparable in both samples. Result is
representative of 3 different experiments.
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Since tyrosine phosphorylation is believed to be required for
activation of the intracellular PLC1, we determined the
phosphorylation levels of this cell surface enzyme. PLC1 was
immunoprecipitated from the membranes and, as an internal control, from
the cytosolic extracts of peripheral T cells. Equal amounts of the
immunoprecipitates were subjected to Western blot analysis using an
anti-phosphotyrosine MoAb (PY-20). The ECL system showed that PLC1
from the membranes was clearly phosphorylated on tyrosine residues,
whereas the cytosolic enzyme displayed weak reactivity due to the
absence of any activation trigger (ie, TCR/CD3 engagement, etc.; Fig
4B). Since it has been convincingly demonstrated that PLC1 resides
in the cytoplasm of resting T cells and is recruited to the membrane
only upon activation,16 it appears unlikely that the
phosphorylated enzyme in the membrane fraction is the cytoplasmic one
recruited to the internal leaflet of the plasma membrane.
We determined next whether the expression of ecto-PLC1 was
correlated with the proliferative state of the cells by extending the
investigations to lymphocytes treated first with an activating agent
like PHA and then with an antiproliferative drug such as natural
interferon beta, whose antireplicative effect on T lymphocytes has
already been reported.17 Treatment of PHA-stimulated
lymphocytes with interferon beta indeed slowed the cell proliferation
within 24 hours of treatment, leading to a progressive reduction of the growth rate over the following 2 days (not shown). Flow cytometric analysis indicated that treatment of the cells with PHA and with PHA
plus interferon did not change either the percentage of
PLC1+ lymphocytes or the distribution between T-cell
subsets (not shown). On the other hand, the confocal microscopic images
showed that PHA alone induced a downregulation of ecto-PLC1
expression (Fig 1B), which was promptly restored by addition of
interferon to the culture medium. This effect was clearly detectable
after 90 minutes of interferon treatment and was still evident after 24 hours (Fig 1C and D). Such results therefore strongly suggest a
relationship between the proliferative state of the cells and ecto-PLC1 expression.
To investigate whether the functional properties of the enzyme were
also modulated by PHA alone and PHA plus interferon treatment, we next
investigated its activity by incubating the cells with [3H]-lyso-PI
and measuring the production of inositol phosphates released in the
medium. [3H]-lyso-PI, due to its high water solubility, forms
micelles that rapidly transfer to the cell membrane. Indeed, the
kinetics of the spontaneous uptake of this lipid by lymphocytes showed
that lyso-PI in the medium plateaued at about 25% within 5 minutes in
all samples, in this way becoming accessible to the ectoenzyme; beyond
5 minutes, the rate of PI transfer was evidently reduced (Fig
5). In all samples, the formation of
radiolabeled inositol phosphates (measured as the aqueous extractable
cpm), which paralleled the disappearance of PI from the medium, was approximately linear over a 5-minute period. Beyond that time, such
production plateaued, weakly increasing between 5 and 30 minutes (Fig
6a). The rapid kinetics of formation of
radiolabeled inositol phosphates in the external medium upon addition
of [3H]-lyso-PI reasonably excludes the possibility that such
production could derive from a sequence of events including (1) lipid
internalization into the cell, (2) its cleavage by an intracellular
phospholipase, and (3) diffusion through the membrane of the hydrolytic
products to the external medium. A parallel incubation of
[3H]-lyso-PI in the medium without cells produced low levels of
radioactivity, further indicating that the radiolabeled production was
due to a cellular phospholipase rather than to unspecific enzymatic
activities in the medium.
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| Fig 5.
Kinetics of [3H]-lyso-PI uptake. Transfer of
[3H]-lyso-PI from donor vesicles to the cell membrane of lymphocytes
was measured by scintillation counting of the incubation medium from
EGTA-containing samples. The 100% value was obtained by measuring an
aliquot of solution of 11.25 µmol/L [3H]-lyso-PI (concentration of
substrate added to the cells). The kinetics of spontaneous uptake of
the lipid by the cells shows that its concentrations in the medium plateaued at about 25% within 5 minutes. Beyond this time, the rate of
lyso-PI transfer was evidently reduced. Data are the mean ± SD of at
least 3 separate experiments.
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| Fig 6.
Formation of water-soluble radiolabeled products in the
absence (a) or presence (b) of EGTA. Assays were performed for the indicated times in the absence or presence of 0.5 mmol/L EGTA. In the
absence of EGTA, production increased linearly at least fivefold over a
5-minute period, plateauing between 5 and 30 minutes. When EGTA was
added to the reactions, recovery of radioactivity dramatically
decreased, indicating its Ca2+ dependency. Data are the
mean ± SD of at least 3 different experiments.
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Scintillation analysis showed that compared with PHA-stimulated cells,
larger amounts of radiolabeled inositol phosphates were produced in
resting and interferon-treated cells, with a main peak in the 90-minute
interferon-treated samples (Fig 6a). These results fit well with the
PLC1 overexpression observed by confocal microscopy in the
corresponding samples (Fig 1).
The reaction appeared to be Ca2+-dependent, since the
presence of EGTA in the incubation medium strongly impaired the
recovery of radioactivity of water-soluble material in all samples (Fig 6b). Such dependency sets this PLC apart from the bacterial and Trypanosoma brucei forms, which do not require Ca2+
for their activity.18,19 Moreover, the almost complete
inactivation of the enzyme observed upon impairment of Ca2+
in the medium indicates that the enzyme also exposes the
Ca2+-binding site(s) outside the cell membrane.
To verify that lyso-PI cleavage occurred through the action of PLC
rather than other enzymes (ie, lysophospholipase or phospholipase A1 or
D), we tested for the specific radiolabeled products of PLC activity,
inositol 1 phosphate (Ins(1)P) and inositol 1:2 cyclic phosphate
(Ins(1:2 cycl)P), by loading the water-soluble material on a small (1 mL) chromatographic Dowex AG1-X8 column.15 By this method,
we were able to identify two main fractions corresponding to Ins(1)P
and Ins(1:2 cycl)P (Fig 7), along with small amounts of
glycero-phospho-myo-inositol, and traces of radioactivity
related to possible contaminants (not shown). Scintillation counting of the chromatographic products showed that, together, Ins(1)P and Ins(1:2
cycl)P appeared particularly high in the resting cells and in
interferon-treated cells (Fig 7). These
results strongly suggest a relationship of ecto-PLC expression and
activity to cell proliferation, and are in accordance with previous
data indicating increased activity of an ecto-PLC in fibroblastic lines
during inhibition of cell growth.12-14
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| Fig 7.
Percentage of radiolabeled inositol phosphates in the
water-soluble material (100%). Lymphocytes in the different
experimental conditions were incubated in a reaction mixture containing
11.25 µmol/L lyso-PI up to 30 minutes. Once the reaction was stopped, the water-soluble products extracted from the incubation medium were
chromatographed over a 1-mL column of Dowex AG 1-X8 resin, and the
fractions were analyzed by scintillation counting. The peak is evident
for Ins(1)P in 90-min interferon-treated samples, while Ins(1:2 cycl)P
constantly increases along the time of treatment and accumulates to
significant concentrations in 24-hour treated samples and in
unstimulated cells. Data are the mean of 3 experiments differing  5%
SD.
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It is known that PLC catalytic activity results in the formation of
diacylglycerol (DAG) other than inositol phosphates, and both of these
second messengers could contribute to the regulation of the
proliferative state of the cells. Since DAG is able to activate protein
kinase C (PKC), we checked for changes in PKC activity in the different
experimental conditions, but the results did not indicate any
substantial differences among the samples (not shown). This finding
thus suggests the inositol phosphates generated by the action of
ecto-PLC as possible regulatory molecules in the signaling route
controlling lymphocyte metabolism. It is well known indeed that inside
the cell, these second messengers, even at low concentration, can
modulate a wide range of cellular activities.20 In
addition, the hypothesis of selective involvement of Ins(1)P and
Ins(1:2 cycl)P in the mechanisms regulating T-lymphocyte behavior might
be taken into account. Indeed, based on the finding of a peak of
radiolabeled Ins(1)P in the 90-minute interferon-treated cells and the
constant increase of Ins(1:2 cycl)P along the time of interferon
treatment, a different involvement of the two molecules could be
supposed: Ins(1)P might be a primary signal for entry in the metabolic
shutdown of proliferation, while Ins(1:2 cycl)P might be required for
establishment and maintenance of the quiescence. The finding that
Ins(1:2 cycl)P accumulates to a significant concentration in 24-hour
interferon-treated cells and in resting cells (ie, unstimulated) is in
line with the suggestion of a distinct regulatory role for these two
compounds. This is also in accordance with previous studies in which
PLC (albeit the intracellular enzyme) is reportedly able to catalyze
the formation of Ins(1)P or Ins(1:2 cycl)P by totally independent
pathways.21 Moreover, it has been previously described that
noncyclic and cyclic inositol phosphates show completely different
kinetics, the former being rapidly metabolized and the latter
displaying a slow turnover.22 These findings allow us to
hypothesize that distinct inositol phosphates, generated by hydrolysis
of the aliquot of PI present on the outer leaflet of the
membrane,23-24 might contribute to regulation of the
lymphocyte metabolic machinery. PLC1 hydrolytic products might thus
be capable of transbilayer diffusion and/or receptor-mediated
endocytosis, with subsequent autocrine or paracrine action on cell
proliferation.
Taken together, the data presented here provide the first evidence of
the presence of a PLC1 on the external surface of some T-cell
subsets, which is apparently involved in the regulation of the
replicative machinery. PLC1+ lymphocytes were
essentially CD8+ and CD45RA+. The structural
and kinetic characterization of the enzyme indicate that (1) it has an
apparent mass of 145 kD and reacts with antibodies directed against
PLC1, (2) it is phosphorylated on tyrosine residues, and (3) its
activity is calcium-dependent.
In terms of cell surface disposition, significant regions of the
enzyme, such as the catalytic active sites, the carboxyl-terminal portion recognized by the anti-PLC1 antibody, and the
calcium-binding site(s), appear to be extracellular domains of the
enzyme and therefore are not directly involved in the mechanisms of
membrane attachment.
This peculiar localization strongly sets this protein apart from the
other known mammalian PLCs, although some biochemical properties are
shared with the intracellular form. Although the possibility of a
synthesis from modified mRNA cannot be completely ruled out, the
relatively rapid increase of the enzyme expression and activity after
interferon treatment would suggest a translocation to the cell surface
from the intracellular stores. If this is the case, phosphorylation
might play an important role in the transfer, since the enzyme at the
external surface appears to be phosphorylated and active. It is
conceivable that conformational changes induced by phosphorylation may
be responsible for the exposure of hydrophobic residues able, in turn,
to anchor the protein to the membrane.
| |
FOOTNOTES |
Submitted June 30, 1997;
accepted December 29, 1997.
Supported by a MURST 60% grant (1995-1996).
Address reprint requests to Sebastiano Miscia, MD,
Istituto di Morfologia Umana Normale, Università G. D'Annunzio,
Via dei Vestini 6, 66100 Chieti, Italy.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
This work is dedicated to the memory of Valerio.
| |
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Abstract
Introduction
Abstract