Biochem. J. (2009) 423, 99–108 (Printed in Great Britain)
99
doi:10.1042/BJ20090687
Specific role of phosphoinositide 3-kinase p110α in the regulation
of phagocytosis and pinocytosis in macrophages
Namiko TAMURA, Kaoru HAZEKI1 , Natsumi OKAZAKI, Yukiko KAMETANI, Hiroki MURAKAMI, Yuki TAKABA, Yuki ISHIKAWA,
Kiyomi NIGORIKAWA and Osamu HAZEKI
Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan
PI3K (phosphoinositide 3-kinase) α has been implicated in phagocytosis and fluid-phase pinocytosis in macrophages. The subtypespecific role of PI3K in these processes is poorly understood. To
elucidate this issue, we made Raw 264.7 cells (a mouse leukaemic
monocyte–macrophage cell line) deficient in each of the classI PI3K catalytic subunits: p110α, p110β, p110δ and p110γ .
Among these cells, only the p110α-deficient cells exhibited
lower phagocytosis of opsonized and non-opsonized zymosan.
The p110α-deficient cells also showed the impaired phagocytosis
of IgG-opsonized erythrocytes and the impaired fluidphase pinocytosis of dextran (molecular mass of 40 kDa).
Receptor-mediated pinocytosis of DiI (1,1 -dioctadecyl-3,3,3 ,3 tetramethylindocarbocyanine perchlorate)-labelled acetylated
low-density lipoprotein and fluid-phase pinocytosis of Lucifer
Yellow (molecular mass of 500 Da) were resistant to p110α
depletion. None of these processes were impaired in cells
lacking p110β, p110δ or p110γ , but were susceptible to a panPI3K inhibitor wortmannin. In cells deficient in the enzymes
catalysing PtdIns(3,4,5)P3 breakdown [PTEN (phosphatase and
tensin homologue deleted on chromosome 10) or SHIP-1 (Srchomology-2-domain-containing inositol phosphatase-1)], uptake
of IgG-opsonized particles was enhanced. These results indicated
that phagocytosis and fluid-phase pinocytosis of larger molecules
are dependent on the lipid kinase activity of p110α, whereas
pinocytosis via clathrin-coated and small non-coated vesicles may
depend on subtypes of PI3Ks other than class I.
INTRODUCTION
regulatory subunit (p85) [10]. These subtypes are thought to be
the major in vivo source of PtdIns(3,4,5)P3 that is produced upon
activation of receptors possessing protein-tyrosine kinase activity
or receptors coupling to Src-type protein-tyrosine kinases [10].
In mammals, there are multiple isoforms of class-IA PI3K
[10]. Different genes encode class-IA catalytic subunits (referred
to as p110α, p110β and p110δ), whereas two genes encode
the associating regulatory subunits (referred to as p85α and
p85β). Class I includes another member, PI3Kγ , which is mainly
expressed in haematopoietic cells. This subtype consists of a
catalytic subunit (p110γ ) and a regulatory subunit (p101) and is
classified as class IB [11,12]. PI3Kγ can be activated by the βγ
subunits of G-proteins and thus mediate the signal from G-proteincoupled receptors [13,14]. Class-II PI3Ks are 170–210 kDa
proteins with an in vitro substrate specificity restricted to PtdIns
and PtdIns4P [15]. Class-III PI3Ks are homologues of the baker’s
yeast (Saccharomyces cerevisiae) Vps34p (vacuolar protein
sorting protein 34) and exclusively phosphorylate PtdIns [16].
PI3K has been implicated in the regulation of phagocytosis,
pinocytosis, membrane ruffling, cytoskeletal organization and
intracellular membrane traffic [1,2]. The involvement of PI3K
in phagocytosis is well characterized, particularly by examining
the Fcγ R (Fcγ receptor)-mediated process in macrophages.
PtdIns(3,4,5)P3 has been shown to localize in the phagocytic cup
at the early stage of phagocytosis before closure of the phagosomal
vacuole [17–19]. The accumulation of PtdIns(3,4,5)P3 is transient,
followed by the accumulation of PtdIns3P concomitant with
Phagocytosis and pinocytosis have substantial roles in the entry
of pathogens into host cells, innate and adaptive immunity
and apoptosis [1–3]. The processes also play a critical part in
foam-cell formation of macrophages to cause development of
atherosclerotic plaques [4]. The mechanism of their regulation
therefore attracts the attention of various medical specialties.
Phagocytosis is a process by which solid particles are
taken up by professional phagocytes such as macrophages,
neutrophils and dendritic cells. Pinocytosis occurs in all cells
and functions to engulf fluid and solutes. The process of
phagocytosis involves specific cell-surface receptors. One group
of the receptors, including mannose receptors and TLRs (Tolllike receptors), interact directly with pathogens by recognizing
the conserved motifs of micro-organisms [5,6]. Another group of
receptors recognize opsonins, host molecules (e.g. antibodies and
complement components) that interact with the surface molecules
of invaders [7,8]. Fluid-phase pinocytosis occurs by at least four
basic mechanisms, namely macropinocytosis, clathrin-mediated
endocytosis, caveolin-mediated endocytosis and clathrin- and
caveolin-independent endocytosis [9].
Mammalian PI3Ks (phosphoinositide 3-kinases) can be
grouped into three major classes (I, II and III) on the basis of
their primary sequences, mechanism of regulation and substrate
specificities [10]. Of the class-I PI3Ks, class-IA subtypes
are heterodimers comprising a catalytic subunit (p110) and a
Key words: Fcγ receptor (Fcγ R), macrophage, phagocytosis,
phosphoinositide 3-kinase α (PI3Kα), pinocytosis, zymosan.
Abbreviations used: DiI, 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyanine perchlorate; DTT, dithiothreitol; EA, IgG-coated SRBC; ERK,
extracellular-signal-regulated kinase; FCS, fetal-calf serum; FcγR, Fcγ receptor; LDL, low-density lipoprotein; LPS, lipopolysaccharide; pAkt(Ser473 ),
Akt/protein kinase B phosphorylated on Ser473 ; pERK1/2(Thr202 /Tyr204 ), ERK 1/2 phosphorylated on Thr202 and Tyr204 ; PI3K, phosphoinositide 3-kinase;
PTEN, phosphatase and tensin homologue deleted on chromosome 10; SHIP, SH2 (Src homology 2)-domain-containing inositol phosphatase; shRNA,
short hairpin RNA; SRBC, sheep red blood cell; TLR, Toll-like receptor.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
100
N. Tamura and others
the sealing of the vacuoles [18,20]. In macropinocytosis, the
PtdIns(3,4,5)P3 concentration has been shown to increase
dramatically in the macropinocytic cup, and PI3K is suggested
to function in the closure of ruffles to form macropinosomes,
as in the case of phagosome formation from the phagocytic cup
[1,21]. Receptor-mediated pinocytosis of soluble ligands through
clathrin-coated vesicles has been shown to be less sensitive to
PI3K inhibitors than macropinocytosis, indicating that the roles
of PI3K in the different types of pinocytosis may not be the same
[21].
Recent studies have suggested that class-I PI3K triggers
initiation of endocytosis [2], but the subtype of class-I PI3K
responsible has not been elucidated. To examine this issue, we
prepared cells deficient in each of the class-I PI3K subtypes and
determined their abilities to take up different sizes of particles
and solutes. We show that the phagocytosis of particles and
the pinocytosis of larger molecules are critically dependent on the
p110α subtype of class-I PI3K. We also show that receptormediated pinocytosis and micropinocytosis of smaller molecules
are not controlled by the class-I subtypes of PI3K.
EXPERIMENTAL
Materials
The sources of materials were as follows: ATP, LPS
(lipopolysaccharide) (Escherichia coli serotype 0111: B4), FITC–
dextran (molecular mass of kDa 40), Lucifer Yellow (molecular
mass of 522 Da) and calyculin-A were from Sigma;
zymosan, FITC–zymosan, anti-zymosan IgG and DiI (1,1 dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyanine perchlorate)labelled acetylated LDL (low-density lipoprotein) were from
Molecular Probes; SRBCs (sheep red blood cells) were from
Japan Lamb; wortmannin was from Kyowa Medex; RPMI
1640 medium was from Invitrogen; the protein assay kit was
from Bio-Rad; cytochalasin-E and latrunculin-B were from
Calbiochem; antibodies against pAkt(Ser473 ) (Akt/protein kinase
B phosphorylated on Ser473 ) and PTEN (phosphatase and tensin
homologue deleted on chromosome 10) were from Cell Signaling
Technology; anti-Akt1/2, anti-p110β and anti-p110γ antibodies
were from Santa Cruz Biotechnology; anti-SRBC antibody was
from InterCell Technologies; anti-pERK1/2(Thr202 /Tyr204 ) [antiERK (extracellular-signal-regulated kinase) 1/2 phosphorylated
on Thr202 and Tyr204 ], anti-p110α, anti-SHIP-1 [anti-(Srchomology-2-domain-containing inositol phosphatase)-1] and
anti-p110δ antibodies were from BD Transduction Laboratories;
phosphatidylserine and phosphatidylinositol were from Matreya;
[γ -32 P]ATP was from New England Nuclear; and Na2 51 CrO4
was from MP Biomedicals. Anti-p85α polyclonal antibody was
prepared by immunizing rabbits with full-length GST (glutathione
transferase)–p85α.
Cells
Female C57/BL6 mice (age 8–12 weeks) were purchased from
Japan SLC. PI3Kγ −/− mice with a C57/BL6 background were
kindly donated by Dr Takehiko Sasaki (Department of Pathology
and Immunology, Akita University, Akita, Japan); peritoneal
macrophages were harvested from these mice. Briefly, mice
were injected intraperitoneally with 2 ml of 3 % thioglycollate
broth. After 3 days, the mice were anaesthetized with ether
inhalation and the peritoneal exudate was harvested by washing
the peritoneal cavity with ice-cold PBS. Cells were seeded (about
5 × 105 cells/well) in 24-well plates and allowed to adhere to
dishes by placing them in an atmosphere of humidified 5 % CO2 at
c The Authors Journal compilation c 2009 Biochemical Society
37 ◦C for 1–2 h in RPMI 1640 medium supplemented with 10 %
(v/v) FCS (fetal calf serum). Non-adherent cells were washed
off with PBS and attached cells were designated macrophages.
The mouse macrophage-like cell line Raw 264.7 was maintained
in RPMI 1640 medium containing 4.5g/l glucose and 10 %
FCS in humidified 5 % CO2 at 37 ◦C. For the determination of
phagocytosis or pinocytosis, cells were cultured in 96-well plates
or 24-well plates respectively. Culture medium were aspirated
off and replenished with incubation buffer (complete RPMI 1640
medium without NaHCO3 , fortified with 20 mM Hepes/NaOH,
pH 7.4). Activities were then determined by incubating cells at
37 ◦C in a water bath under ambient conditions.
Animal experiments were performed with approbation by
the institutional ethics committee of Hiroshima University,
in accordance with the Standards Relating to the Core and
Management of Experimental Animals in Japan.
Western blotting
Cells were washed with PBS and lysed in 50 μl of lysis
buffer containing 25 mM Tris/HCl (pH 7.4), 0.5 % Nonidet P40,
150 mM NaCl, 1 mM Na3 VO4 , 1 mM EDTA, 0.1 % BSA, 20 mM
NaF, 1 mM PMSF, 2 μM leupeptin, 20 μM p-amidino-PMSF
and 1 mM DTT (dithiothreitol). Cell lysates were centrifuged
at 20 000 g for 10 min. Protein concentrations in the resultant
supernatants were determined with the Bio-Rad protein assay kit.
Total cell lysates were mixed with 10 μl of 5× sample buffer
[62.5 mM Tris/HCl, pH 6.8, 1 % (w/v) SDS, 10 % (v/v) glycerol,
5 % (v/v) 2-mercaptoethanol and 0.02 % Bromophenol Blue] and
heated at 100 ◦C for 3 min. Proteins (100 μg/lane) were separated
by SDS/PAGE. They were then transferred by electrophoresis on
to PVDF membranes (Millipore). The membranes were blocked
in 5 % (w/v) dried skimmed milk powder and incubated with the
appropriate antibodies. Associated antibodies were detected by
enhanced chemiluminescence (PerkinElmer).
Phagocytosis of zymosan
FITC-labelled zymosan particles were mixed with a 4-fold excess
of unlabelled zymosan to expedite counting. Particles were then
opsonized (or not opsonized) with normal mouse serum or with
anti-zymosan IgG at 37 ◦C for 30 min before use. Subconfluent
monolayers of Raw 264.7 cells (104 /well of a 96-well plate) were
treated with or without inhibitors at 37 ◦C for 15 min, after which
5 × 105 zymosan particles were added and the mixture incubated
at 37 ◦C for a further 60 min. Phagocytosis was stopped by the
addition of ice-cold PBS. Cells were then washed three times
with PBS, fixed with 4 % (v/v) paraformaldehyde for 10 min
at room temperature (23–27 ◦ C), and finally rinsed with PBS.
Fluorescence images (excitation at 488 nm, emission at 525 nm)
and phase-contrast images of at least 100 macrophages from three
randomly selected fields were taken using a confocal microscope.
The mean numbers of ingested zymosan particles were expressed
as particles/one Raw 264.7 cell.
Preparation of IgG-coated red blood cells and measurement
of phagocytosis
SRBCs were labelled with 51 Cr as described previously [22].
EAs (IgG-coated SRBCs) were prepared by incubating labelled
cells with rabbit anti-SRBC antibody at 37 ◦C for 10 min in
5 mM veronal buffer (pH 7.5) supplemented with 0.1 % gelatin,
75 mM NaCl, 0.15 mM CaCl2 , 0.5 mM MgCl2 and 10 mM
EDTA, followed by incubation on ice for 15 min. EAs were
washed three times with GVB [5 mM veronal (sodium barbital)
buffer, pH 7.5, supplemented with 0.1 % gelatin, 75 mM NaCl,
Differential regulation of endocytosis by PI3Kα
Table 1 Oligonucleotides with sequences targeting p110α, p110β, p110γ ,
p110δ, PTEN or SHIP-1
Oligonucleotide target
p110 isoforms
α
β
Sequence
γ
δ
5 -GAACAAGGGCGAGATATAT-3
5 -GTGGAATAAACTTGAAGAT-3
5 -GAAGCAGCCGTGTTATTAT-3
5 -GCAATGTGGAACAGATGAA-3
5 -CACTGAATGACTTTGTGAA-3
PTEN
5 -GAACAATATTGATGATGTA-3
SHIP-1
5 -GGAATGAAATGCTTGAAGA-3
0.15 mM CaCl2 and 0.5 mM MgCl2 ] and finally suspended in
short-term incubation buffer. Binding and phagocytosis of EAs
were measured as reported previously [22]. Monolayers of Raw
264.7 cells (2 × 105 cells/well of a 24-well plate) were incubated
with 51 Cr-labelled EAs (2 × 107 cells) at 37 ◦C for the indicated
periods of time. Monolayers were washed three times with PBS
to remove unbound EAs, before brief exposure to 0.1 ml of
hypo-osmotic PBS (5-fold-diluted). The radioactivity released
into the supernatant during hypo-osmotic shock was measured
for the amount of EAs bound on the surface of the phagocytes.
Monolayers were washed a further three times with PBS and
finally solubilized in 0.5 % Triton X-100. The radioactivity in
the solution was measured for the amount of engulfed EAs.
The number of EAs was calculated from the radioactivity and
expressed as incorporated or bound EAs/one Raw 264.7 cell.
Fluid-phase pinocytosis assay
FITC-labelled dextran (0.5 mg/ml), Lucifer Yellow (0.5 mg/ml)
and DiI-acetylated LDL (8 μg/ml) were used to measure
pinocytosis. Cells (5 × 105 cells/well of a 12-well plate) were
incubated in the presence or absence of inhibitors, and the labelled
targets added. After further incubation for various intervals, the
medium were aspirated. Cells were washed twice with 0.1 %
BSA/PBS, three times with PBS, and finally solubilized in 150 μl
of 0.1 % Triton X-100/PBS. Fluorescence intensity was measured
at an excitation wavelength of 488 nm and an emission wavelength
of 520 nm for FITC–dextran and Lucifer Yellow, and at an
excitation wavelength of 544 nm and an emission wavelength
of 590 nm for DiI-labelled acetylated LDL.
RNA interference
Oligonucleotides with the sequence targeting p110α, p110β,
p110γ , p110δ, PTEN or SHIP-1 were cloned into the pH1 vector
downstream of the H1 RNA promoter as described in [23,24] to
express siRNA (small interfering RNA) hairpins. The sequences
targeted are shown in Table 1. For each of the targeted sequences,
a pair of oligonucleotides were synthesized with sequences 5 -CCC(X)19 TTCAAGAGA(Y)19 TTTTTGGAAA-3 and 5 -CTAGTTTCCAAAAA(Y)19 TCTCTTGAA(X)19 GGGTGCA, where (X)19
is the coding sequence and (Y)19 is the complementary sequence.
The oligonucleotide pair were annealed and ligated downstream
of the H1 RNA promoter at the PstI and XbaI sites of the pH1
vector. The vectors were transfected into Raw 264.7 cells [(5–
10) × 106 cells] at 250 V/950 μF (Gene Pulser II; Bio-Rad). When
p110β was targeted, two shRNA (short hairpin RNA) probes
with different sequences were transfected simultaneously to fully
inhibit protein expression. At 24 h after transfection, puromycin
101
(5 μg/ml) was added to the cells for selection, and the incubation
continued for several days. Resistant colonies (50–100) were
replated on 96-well plates, to give 0.5–1.0 cells/well, and cultured
for additional weeks to obtain monoclonal PI3K-deficient cells.
To determine the efficiency of gene silencing, total RNA was
isolated with RNeasy (Qiagen) and mRNA was quantified by
reverse transcription–PCR. Control cells were prepared in the
same way with pH1 vector containing a 400 bp stuffer sequence
instead of the probe sequence.
PI3K activity assay
Anti-p85 immunoprecipitates were assayed for their PI3K
activities. The cell lysate was precleared with preimmune IgG
and Protein G–Sepharose at 4 ◦C for 1 h, and subjected to
immunoprecipitation with anti-p85 antibody. Immunoprecipitates
were washed twice with lysis buffer, twice with 40 mM Tris/HCl
(pH 7.4)/1 mM DTT/0.5 M LiCl, and twice with 40 mM Tris/HCl
(pH 7.4)/1 mM DTT/100 mM NaCl. Aliquots (equivalent to
2 × 106 cells) of the immunoprecipitates were suspended in 0.1 ml
of a reaction mixture consisting of 40 mM Tris/HCl, pH 7.4,
0.5 mM EGTA, 0.2 mM PtdIns, 0.2 mM phosphatidylserine,
5 mM MgCl2 and 0.2 mM (1 μCi) of [γ -32 P]ATP. The reaction
was allowed to proceed at 37 ◦C for 15 min before termination
by the addition of 20 μl of 8 % (w/v) HClO4 and 0.45 ml of
chloroform/methanol (1:2, v/v). After vigorous stirring, 0.15 ml of
chloroform and 0.15 ml of 8 % HClO4 were added to the mixture
to separate the organic phase. The organic phase was washed
with chloroform-saturated 0.5 M NaCl containing 1 % HClO4
and evaporated to dryness. The extract was dissolved twice in
15 μl of chloroform/methanol (9:1, v/v) to be spotted on to a
silica-gel plate (Silica Gel 60; Merck). The plate was developed
in chloroform/methanol/28 % NH4 OH/water (70:100:25:15 by
vol.), dried and visualized for radioactivity in the phosphoinositide
fraction using a Fuji BAS3000 imaging analyser.
RESULTS
Involvement of class-I PI3Ks in phagocytosis
Figure 1(A) shows microscopic images of Raw 264.7 cells given
IgG-opsonized zymosan. The phase-contrast image of control
cells indicates that multiple particles were engulfed by one cell.
Fluorescence micrographs and the merged image show that a
part of the engulfed particles was labelled by fluorescence. This
is because zymosan particles were given as a mixture of FITClabelled and non-labelled forms for convenience in quantification.
When cells were treated with wortmannin before zymosan
addition, uptake was minimal and particles were observed at
the outer surface of cells. Because the micrographs were taken
after the cells had been washed repeatedly, these particles were
probably recognized by cell-surface receptors, but were not
engulfed by the cells. In Figure 1(B), the number of the fluorescent
particles within cells was counted using merged images. Counting
was corrected by the content of the labelled form in the zymosan
mixture to estimate the endocytotic activity as particles per
one cell. In this experiment, zymosan particles were used in
non-opsonized, IgG-opsonized or C3bi [a fragment of the third
component of complement (C3)]-opsonized forms. Wortmannin
inhibited zymosan uptake in a dose-dependent manner, regardless
of whether the particles were opsonized or not. The inhibition of
phagocytosis was observed when LY294002 was used as a PI3K
inhibitor with the maximal effect at 100 μM (Figure 1C).
The results of the pharmacological analysis described
above agreed well with those from previous studies [22,25] and
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N. Tamura and others
Figure 2
cells
Enhancement of Fcγ R-mediated phagocytosis in SHIP-1-deficient
(A) Lysates from SHIP-1-deficient and control (Cont.) Raw 264.7 cells were analysed by Western
blotting with anti-SHIP-1 or anti-β-actin antibodies. (B and C) SHIP-1-deficient cells (䊊) or
control (Cont.) cells (䊉) were treated with increasing concentrations of wortmannin for 15 min
and then for 60 min with the addition of EAs. The number of EAs within the cells (phagocytosis,
B) and on the outer surface of the cells (binding, C) was determined as described in the
Experimental section. Similar results were obtained in a repeated experiment.
Figure 1 Inhibitory effect of PI3K inhibitors on the phagocytosis of zymosan
particles
(A) Raw 264.7 cells were incubated with wortmannin or vehicle for 15 min and for a further
60 min with the addition of IgG-opsonized (op) zymosan. Zymosan particles were given as
a mixture of FITC-labelled and non-labelled forms. Fluorescent, phase-contrast and merged
images are shown. (B) Cells were incubated with wortmannin or vehicle for 15 min and for
a further 60 min with the addition of non-opsonized (Non-op), serum-opsonized (C3bi-op) or
IgG-opsonized (IgG-op) zymosan. The number of fluorescent particles was counted on merged
images, and the number of zymosan particles within cells calculated on the basis of the content
of the FITC-labelled form. (C) Cells were incubated with LY294002 or vehicle for 15 min and for
a further 60 min with the addition of IgG-opsonized zymosan. The number of zymosan within
cells was quantified as in (B). Representative data from the experiments repeated three times (A
and B) or twice (C) are shown.
suggested that the lipid products of PI3Ks play a critical
role in phagocytosis. It is known that class-I PI3Ks possess
protein kinase activity, which is also inhibited by PI3K inhibitors
c The Authors Journal compilation c 2009 Biochemical Society
[10]. We therefore next examined whether treatments that
perturb the metabolism of PI3K lipid products have an effect
on phagocytosis. For this purpose, we prepared Raw 264.7 cells
lacking SHIP-1 or PTEN using an shRNA technique. Uptake of
IgG-opsonized SRBCs by these cells was then examined.
SHIP is a 5 -phosphatase that metabolizes the in vivo product of
class-IA PI3Ks, PtdIns(3,4,5)P3 , to produce PtdIns(3,4)P2 [26].
Introduction of the shRNA probe abolished the protein expression
of SHIP-1 (Figure 2A). Fcγ R-mediated phagocytosis of IgGopsonized SRBCs doubled in SHIP-1-deficient cells (Figure 2B),
suggesting that the increased level of PtdIns(3,4,5)P3 enhances the
activity. The increased number of Fcγ Rs is not the mechanism of
this enhancement, because the binding of IgG-opsonized SRBCs
on the cell surface in these cells was unchanged (Figure 2C). This
was confirmed by the finding that the uptake of non-opsonized
zymosan engulfed through binding to mannose receptors and
TLRs instead of Fcγ R was again increased in these cells
(results not shown). PI3K inhibition by wortmannin decreased
the phagocytosis of control and SHIP-1-deficient cells in a similar
dose-dependent manner (Figure 2B), but had no effect on the cellsurface binding of SRBCs (Figure 2C).
PTEN is another enzyme metabolizing PtdIns(3,4,5)P3 that
catalyses the 3 -dephosphorylation to produce PtdIns(4,5)P2
[27]. The knockdown of PTEN markedly increased the rate
Differential regulation of endocytosis by PI3Kα
Figure 4
Figure 3
cells
Enhacement of Fcγ R-mediated phagocytosis in PTEN-deficient
(A) Lysates from PTEN-deficient and control (Cont.) Raw 264.7 cells were analysed by Western
blotting with anti-PTEN or anti-Akt antibodies. (B and C) PTEN-deficient (PTEN) cells (䊊)
or control (Cont.) cells (䊉) were incubated for various intervals with the addition of EAs. The
number of EAs within the cells (phagocytosis, B) and on the outer surface of the cells (binding,
C) was determined as described in the Experimental section. Similar results were obtained in a
repeated experiment.
of Fcγ R-mediated phagocytosis (Figure 3B). The increase was
more remarkable than that observed in SHIP-1-deficient cells
(Figure 2B), probably because of the different spatial distribution
of these enzymes and the presence of 5 -phosphatase isozymes
[28–30]. The amount of IgG-opsonized SRBCs on the cell surface
decreased slightly in PTEN-deficient cells, presumably because
of the higher uptake of target particles (Figure 3C).
Specific role of p110α in phagocytosis
The results described above, together with a previous report
showing the specific localization of PtdIns(3,4,5)P3 in phagocytic
cups [1], suggested that the lipid product of class-I PI3K has a
substantial role in Fcγ R-mediated phagocytosis. To determine
the PI3K subtype responsible for this process, Raw 264.7 cells
deficient in p110α, p110β, p110δ or p110γ were prepared. Raw
264.7 cells were transfected with an shRNA probe targeting each
of the p110 catalytic subunits by electroporation. Cells were
cultured in the presence of puromycin for about 2 weeks; resistant
cells were cloned and expanded for use. Figure 4(A) shows the
result of Western-blotting analysis of a series of p110α-depressed
clones. Clones 2.4 and 2.1 showed minimum expression of p110α,
and thus these clones were used in the experiments described
below. The p110α-deficient cells showed normal expression of
the p85 regulatory subunit of PI3K and the catalytic subunits
other than p110α (Figure 4B). The p110β-, p110δ- or p110γ -
103
Preparation of cells deficient () in each PI3K catalytic subunit
Raw 264.7 cells were transfected with 20 μg of an shRNA vector targeting each of p110α
(p110α), p110β (p110β), p110δ (p110δ) and p110γ (p110γ ) or control vector
(Cont.) by electroporation. Cells resistant to puromycin were selected and cloned as described
in the Experimental section. (A) Cell lysates from cloned cells were analysed by Western blotting
with anti-p110α or anti-β-actin antibodies. (B) Total lysates or anti-p85 immunoprecipitates
(IP) were analysed by Western blotting with the antibodies against p85, p110α, p110β, p110δ,
p110γ or PTEN.
deficient cells showed a specific decrease of the targeted subunit
(Figure 4B).
Prepared cells were first examined for their ability to engulf
IgG-opsonized (Figure 5A), C3bi-opsonized (Figure 5B) and
non-opsonized (Figure 5C) zymosan particles. The phagocytosis
of these particles was impaired markedly in p110α-deficient
cells. Microscopic images of p110α-deficient cells given FITClabelled IgG-opsonized zymosan showed that the particles were
observed sometimes on the surface of, but merely within, the
cells (Figure 5D), as seen in wortmannin-treated cells (see
Figure 1A). The decreased phagocytosis in p110α-deficient
cells was also observed when uptake of IgG-opsonized SRBCs
was examined (Figures 5E and 5F). The phagocytosis of
the particles was not decreased in cells deficient in p110β,
p110δ and p110γ (Figures 5A–5C and 5F). The irrelevance of
p110γ in the processes was confirmed by the experiment with
macrophages from p110γ −/− mice (right panels of Figures 5A–
5C). The effects of the p110α deficiency described above
were observed similarly when another targeting sequence (5 GCAACCTATGTAAATGTAA-3 ) was used to silence p110α
expression (results not shown).
Roles of p110α in macropinocytosis, micropinocytosis and
receptor-mediated endocytosis
Macrophages are endocytic cells known to exhibit, in addition
to the phagocytosis of solid particles, fluid-phase pinocytosis
and receptor-mediated endocytosis of soluble ligands. We next
examined whether these processes deteriorate in p110α-deficient
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Figure 5
N. Tamura and others
Decreased phagocytosis in p110α-deficient cells
(A–C, left-hand panels) Control cells and cells deficient in p110α, p110β, p110δ or p110γ (α–γ ) were incubated with IgG-opsonized zymosan (A), serum (C3bi)-opsonized zymosan (B) or
non-opsonized zymosan (C) for 60 min. The amounts of engulfed zymosan were quantified as described in Figure 1 and the amount of zymosan taken up by control cells during 60 min incubation
is expressed as 100 %. Each bar represents the mean +
− S.D. for three independent experiments (*P < 0.05; **P < 0.01). wort, wortmannin. (A–C, right-hand panels) Macrophages from wild-type
(WT) or p110γ -knockout (p110γ −/− ) mice were evaluated for phagocytosis. Results shown are the means for two separate experiments. (D) Fluorescence, phase-contrast and merged images of
control and p110α-deficient cells given FITC-labelled IgG-opsonized zymosan. (E) p110α-deficient cells (䊉) or control cells (䊊) were incubated for various periods of time with the addition of
EAs. After hypo-osmotic lysis of extracellular EAs, the number of EAs within the cells was determined as described in the Experimental section. (F) Control cells and cells deficient in p110α, p110β,
p110δ or p110γ were incubated for 60 min with the addition of EA. The numbers of cell-surface (binding) and intracellular (phagocyotosis) EAs were determined as described in the Experimental
section. The results are shown as percentages of those for control cells.
cells. The uptake of FITC-labelled dextran (molecular mass of
40 kDa) and Lucifer Yellow (molecular mass of 500 Da) was
examined because it has been shown that different mechanisms
function in the pinocytosis of different ligands depending on their
size [31].
Nocodazole prevented the uptake of these soluble ligands
as well as that of IgG-opsonized particles, indicating the
indispensable role of microtubule re-organization in these
processes (Figure 6). Inhibition of actin assembly by cytochalasinE or latrunculin-B did not decrease the pinocytosis, despite the fact
that the treatment markedly impaired phagocytosis (Figure 6). The
uptake mechanism of soluble ligands was therefore considered to
c The Authors Journal compilation c 2009 Biochemical Society
be different from that of solid particles, in agreement with the
results of previous studies [32]. In spite of this difference, FITC–
dextran uptake was severely inhibited by wortmannin (Figure 7A),
as was phagocytosis. FITC–dextran uptake was decreased by the
depletion of p110α, but was not affected by the depletion of other
subtypes (Figure 7B).
The uptake of a smaller molecule, Lucifer Yellow, was
similar to that of FITC–labelled dextran in its sensitivity to
nocodazole, cytochalasin-E and latrunculin-B (Figure 6). The
uptake mechanism may be different from that of the larger
molecule, because the volume of engulfed liquid containing
Lucifer Yellow was calculated to be 1.3 μl/h per 106 cells, which
Differential regulation of endocytosis by PI3Kα
105
Figure 6 Effect of inhibitors of cytoskeleton assembly on phagocytosis and
pinocytosis
Raw 264.7 cells were preincubated with nocodazole (1 μM), latrunculin-B (100 μM)
cytochalasin-E (10 μM) or vehicle (control) for 15 min. IgG-opsonized (op) SRBC,
IgG-opsonized zymosan, dextran or Lucifer Yellow was added and the mixture incubated for a
further 60 min. The amount of each ligand taken up by control cells during 60 min incubation
is expressed as 100 %. Results are means for two independent experiments.
was quite different from that containing dextran (0.1 μl/h per
106 cells). The uptake of Lucifer Yellow was not affected by
the depletion of p110α, p110β, p110δ or p110γ (Figure 8A).
Despite this apparent independence of class-IA PI3K subtypes,
pinocytosis was partially inhibited by wortmannin (Figure 8B).
The target molecule of wortmannin functioning in this system
may be class-II or class-III PI3K.
Foam-cell formation in macrophages is an important process
in the development of atherosclerotic plaques. LDL uptake in
this process has been reported to occur through actin-dependent
macropinocytosis mediated by Rho GTPase and PI3K signalling
[4,21]. We tested p110α involvement in this process using DiIlabelled acetylated LDL. Similar to the case of Lucifer Yellow,
wortmannin partially inhibited LDL uptake but the defect in
p110α did not affect uptake (Figure 9).
Lower steady-state level of PI3K activity in quiescent
p110α-deficient cells
The results detailed above suggested that p110α is the sole
specific player among class-IA PI3K subtypes that regulates
the phagocytosis of solid particles and the pinocytosis of larger
molecules. One possible explanation for the results is that
activation of every PI3K subtype is impaired in p110α-deficient
cells by a non-specific effect of the shRNA probe. We therefore
examined the activation of a downstream target of class I PI3K
subtypes, Akt (also called protein kinase B), in response to LPS
stimulation. LPS-induced Akt phosphorylation was not impaired,
but markedly increased in p110α-deficient cells (Figure 10A), in
agreement with our previous report [33]. The activation of ERK,
which is not impaired by PI3K inhibitors in these cells (results
not shown), was unaffected in p110α-deficient cells (Figure 10A),
suggesting that these cells do not suffer non-specific loss of
viability.
We next determined the PI3K activity in the cells deficient
in each of the p110 subunits. Lysates from quiescent cells were
used for immunoprecipitation with the antibody against the p85
regulatory subunit. PI3K activity associated with the precipitates
was determined in a cell-free system. PI3K activity was extremely
low in the p110α-deficient cells compared with the other cells
(Figure 10B), indicating that basal PI3K activity in the quiescent
Figure 7
Decreased pinocytosis of dextran in p110α-deficient cells
(A) Raw 264.7 cells were preincubated with wortmannin (䊉) or vehicle (control) (䊊) for 15 min,
after which FITC-labelled dextran was added, followed by further incubation for various periods
of time. (B) Raw 264.7 cells deficient in each of the p110 catalytic subunits were incubated
with FITC-labelled dextran. In (A) and (B), the amount of dextran taken up by control cells
during 60 min incubation is expressed as 100 %. Each point indicates the mean +
− S.D. for three
independent experiments.
state is mainly governed by the α-subtype. This basal activity may
be critical in determining the steady-state levels of phagocytosis
and macropinocytosis.
DISCUSSION
Four isoforms of class-I PI3K catalytic subunit, termed p110α,
p110β, p110γ and p110δ, have been identified. Although p110α
and p110β are widely expressed and share a high degree of
sequence homology, growing evidence suggests a difference in
their physiological roles. This difference is shown by the fact that
targeted disruption of p110α or p110β causes death at an early
embryonic stage [34]. Studies (including those with subtypeselective inhibitors) are accumulating detailed information on
their functional difference [35,36]. In macrophages, we have
reported that p110β has a critical role in LPS-induced activation of
Akt and negative regulation of nitrite production [33]. The present
study indicated the specific function of p110α in two processes
c The Authors Journal compilation c 2009 Biochemical Society
106
N. Tamura and others
Figure 9
Irrelevance of p110α on the pinocytosis of acetylated LDL
Control or p110α-deficient cells were preincubated with (+) or without (−) 0.1 μM wortmannin
(wort), added with DiI-labelled acetylated LDL (LDL) and incubated for a further 60 min. Results
are means +
− S.D. for three independent experiments.
Figure 8
Irrelevance of p110α on the pinocytosis of Lucifer Yellow
(A) Raw 264.7 cells deficient in each of the p110 catalytic subunits were incubated with Lucifer
Yellow. Each point represents the mean +
− S.D. for three independent experiments. (B) Control
or p110α-deficient cells were preincubated with wortmannin (wort; black symbols) or vehicle
(white symbols) for 15 min, after which Lucifer Yellow was added, followed by further incubation
at various intervals. Each point represents the mean for two independent experiments. In (A)
and (B) the amount of Lucifer Yellow taken up by control cells during 60 min incubation is
expressed as 100 %.
of endocytosis: phagocytosis of solid particles and pinocytosis of
larger molecules.
Biochemical studies have identified the functional difference
between p110α and p110β. The activity of p110β is, whereas
that of p110α is not, enhanced by the βγ subunits of G-proteins
[24,37]. It has also been reported that p110α is more active than
p110β at higher substrate concentrations, whereas the opposite
is true at lower substrate concentrations, indicating that p110α
has a higher K m and V max than p110β [38]. The authors of the
latter study suggested that p110α works best in areas of high
substrate density, whereas p110β would function better in areas of
membranes containing low levels of substrate [38]. The difference
in kinetic parameters may explain (at least in part) why p110α
has a preferential role in PI3K-dependent endocytic processes,
because the local level of phosphoinositides is reported to be high
in membranes of phagocytic and macropinocytic cups [1,39]. The
higher V max value of p110α may also explain why the in vitro
PI3K activity from p110α-deficient cells is markedly lower than
c The Authors Journal compilation c 2009 Biochemical Society
Figure 10
In-vivo and in-vitro PI3K activities of p110α-deficient cells
(A) Control or p110α-deficient cells were stimulated with 100 ng/ml LPS or 50 nM calyculin-A
(CA) for 10 min. Whole-cell lysates were analysed by Western blotting with the antibody against
pERK1/2(Thr202 /Tyr204 ) (p-Erk1/2), pAkt(Ser473 ) (p-Akt), or Akt (Total Akt). Similar results were
obtained with another clone. (B) Lysates from control cells or cells deficient in each of the p110
catalytic subunits were subjected to immunoprecipitation with the antibodies against p85. PI3K
activity associated with the immune complex was determined as described in the Experimental
section. (C) The result in (B) was quantified by using an imaging analyser. The activity observed
in the control cells is expressed as 100 %. Results are means +
− S.D. for three independent
experiments.
Differential regulation of endocytosis by PI3Kα
that from other cells (Figure 10C), despite the expression of the
p85 regulatory subunit being not quite different (Figure 4B). The
apparent decrease in PI3K activity in p110α-deficient cells does
not impair every PI3K function in the cells, because LPS-induced
activation of Akt (a p110β-dependent process) did not deteriorate
(Figure 10A).
Several lines of evidence have indicated the role of
PtdIns(3,4,5)P3 in the Fcγ R-mediated process of phagocytosis
[17–19]. Overexpression of the 3 -phosphatase PTEN or the
5 -phosphatase SHIP-1 is reported to inhibit phagocytosis
[40–43]. Macrophages from SHIP-1 knockout mice exhibit
increased uptake of IgG-opsonized SRBC [41]. In the present
study, we confirmed the physiological role of lipid phosphatases
(Figures 2 and 3). The enhancement in phagocytosis (fold increase
above that seen in control cells) by specific knockdown was
greater in PTEN-deficient cells than in SHIP-1-deficient cells.
One possible explanation for the difference is that PTEN is a
predominant catalyst of PtdIns(3,4,5)P3 breakdown in Raw 264.7
cells. The difference may be best explained by their different roles
in the process of phagocytosis; recent studies have demonstrated
that these enzymes show temporally and spatially different
behaviour in phagosomes [28]. The existence of isozymes such as
SHIP-2 may also explain the minor effect of SHIP-1 deficiency
[30].
The p85 regulatory subunit has been shown to be recruited
to the phagocytic cups during Fcγ R-mediated phagocytosis
[28]. This suggests a role for class-IA PI3Ks in increasing
the local concentration of PtdIns(3,4,5)P3 at the site where
an opsonized particle is taken up. In the present study, we
showed that one subtype of class-IA PI3K (p110α), not classIB PI3K p110γ , which is expressed in haematopoietic cells
specifically and abundantly, is a critical determinant of Fcγ Rmediated phagocytosis. We also showed that the uptake of nonopsonized zymosan particles mediated by TLRs and mannose
receptors was impaired in p110α-deficient cells (Figure 5). One
possible function of p110α in these processes is the stimulation of
extension of actin-rich pseudopodia along the engulfed particles.
This possibility may not be the case, because PI3K inhibitors
have been reported not to influence this process [21]. The
action of p110α on processes other than pseudopodia extension
is supported by the present study: the pinocytosis of dextran
molecules, which was not affected by the inhibitors of actin
assembly (Figure 6), was also impaired in p110α-deficient cells.
During the course of the present study, Lee et al. [44] reported
that p110α is required for the uptake of IgG-opsonized and
non-opsonized zymosan in PMA-differentiated THP-1 cells,
although the roles of other isoforms were not examined.
The result is in good agreement with the present study with
Raw 264.7 cells, but was in sharp contrast with the result
obtained for bone-marrow-derived mouse macrophages [45].
The latter study indicated that microinjection of an inhibitory
antibody against p110β (and to a lesser extent an antip110δ antibody) impaired, whereas anti-p110α antibody had no
effect on, Fcγ R-mediated phagocytosis. Distinct roles of PI3K
isoforms in different cell types have been observed in several
cell functions. For example, p110α is a main player in insulin signalling in several cell lines, whereas this is not true in
other cells in which p110β and p110δ show redundant functions
[46]. Colony-stimulating-factor-1-induced proliferation of bonemarrow macrophages is critically dependent on p110δ, but that
of murine macrophage BAC1.2F5 cells is dependent on p110α
[47]. One simple explanation for the difference is that PI3K
isoforms have redundant functions, but their relative expression
level is different between cells. This may not be true in the latter
case at least, because inhibition of p110δ attenuates the PI3K-
107
dependent chemotaxis in both cell lines [47]. In Raw 264.7 cells,
LPS-induced activation of Akt is not impaired in p110α-deficient
cells (Figure 10A). It is of interest to consider that the kinetic
parameters of PI3K isoforms are differently influenced by many
factors in cells (e.g. composition of membrane phospholipids,
presence of co-signals).
Engulfment of soluble ligands is known to occur through
multiple mechanisms, including macropinocytosis, clathrinmediated endocytosis, caveolin-mediated endocytosis, and
clathrin- and caveolin-independent endocytosis. The role of
phosphoinositides in the regulation of these processes is
incompletely understood. We observed that a pan-PI3K inhibitor
wortmannin inhibits, although partly, the uptake of Lucifer Yellow
(Figure 8B) and acetylated LDL (Figure 9), in agreement with
previous studies [21]. Cells lacking class-IA PI3Ks engulfed these
ligands normally (Figures 8A and 9). Because wortmannin is
known to inhibit a broad array of enzymes, its effects on cells is
not necessarily due to changes in phosphoinositide turnover. The
possibility that PI3Ks other than class-I subtypes are operating in
these processes cannot be excluded. Alternatively, deletion of a
single subtype may not have an effect because of the functional
redundancy of class-IA subtypes in these types of endocytosis.
In the present study we have shown that shRNA-based
knockdown of class-IA PI3K is a useful approach to examine
its roles in endocytosis. Further study targeting the subtypes of
class-II PI3K, class-III PI3K and the regulatory subunits of classIA PI3K may help us to understand the mechanisms of p110αindependent processes of endocytosis and to identify the target of
wortmannin in the processes.
AUTHOR CONTRIBUTION
Namiko Tamura and Kaoru Hazeki designed the experiments, performed most of the
experiments and wrote the manuscript. Natsumi Okazaki prepared p110δ-knockdown
cells. Yukiko Kametani assisted in the pinocytosis assay. Hiroki Murakami prepared
SHIP-1 knockdown cells and performed the experiments described in Figure 2. Yuki
Takaba prepared PTEN-knockdown cells and performed the experiments described in
Figure 3. Yuki Ishikawa assisted in the pinocytosis assay. Kiyomi Nigorikawa discussed
the manuscript with us. Osamu Hazeki designed experiments, critically analysed the data
and wrote the manuscript.
ACKNOWLEDGEMENTS
We thank Dr Takehiko Sasaki (Department of Pathology and Immunology, Akita University,
Japan) for p110γ knockout mice, and Dr Norimitsu Inoue (Osaka Medical Center for
Cancer and Cardiovascular Diseases, Osaka, Japan) for shRNA vectors. We also thank the
Analysis Center of Life Science, Hiroshima University, Hiroshima, Japan, for use of their
facilities.
FUNDING
This work was supported by a Grant-in-Aid (Kakenhi) from the Japan Society for the
Promotion of Science [grant number 20590060].
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Received 5 May 2009/25 June 2009; accepted 15 July 2009
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