From the Department of Internal Medicine, Division of
Gastroenterology and Metabolism, Philipps-University, D-35033
Marburg, Germany, the ¶ Research Division, Joslin Diabetes Center
and Department of Medicine and the
Department of Signal
Transduction, Harvard Medical School, Boston, Massachusetts 02215, and the ** Department of Molecular Biology and
Biochemistry, University of California, Irvine, California 92697
Received for publication, August 19, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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The p85 Phosphoinositide 3-kinase
(PI3K)1 generates
phosphorylated phosphoinositides (PI), which serve as crucial
second messengers for a wide range of biological functions including
mitogenesis, survival, differentiation, and cytoskeletal organization.
PI3Ks are divided in three major classes according to substrate
specificity, amino acid sequence, and homology of their lipid kinase
domains. Class IA PI3K phosphorylates phosphatidylinositol
(PtdIns), PtdIns 4-phosphate, and PtdIns 4,5-bisphosphate and are
active as heterodimers consisting of regulatory and catalytic subunits.
Activation of class 1A PI3K occurs by receptor-tyrosine kinases like
the insulin-like growth factor-1 (IGF-1) receptor either by direct
binding to tyrosine-phosphorylated pYMXM and
pYXXM motifs of the IGF-1 receptor There are multiple regulatory and catalytic subunits of class 1A PI3K.
These regulatory subunits are derived from three different genes and
can be classified according to their molecular structure. The
full-length regulatory subunits are derived from distinct genes, the
Pik3r1 (p85 Recent studies indicate a complex regulation of PI3K activity by
regulatory isoforms. In the classical model, p85 In summary, current models of PI3K activation suggest a dual role for
p85 Materials--
Silica-gel thin layer chromatography plates were
obtained from Merck (Darmstadt, Germany), protein G-agarose was from
Santa Cruz Biotechnology (Santa Cruz, CA). Nitrocellulose paper
(Optitran BA-S85) was from Schleicher and Schuell (Keene, NH),
[ Cell Culture--
ES cells lines were grown as
previously described (19) in high glucose Dulbecco's modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum, 10 mM HEPES, 50 µM BrdUrd Incorporation--
Cells were seeded at a density of
3 × 103 in 96-well plates, grown for 24 h in
regular medium, washed once with 10 mM phosphate-buffered saline (pH 7.4) and subsequently starved for 24 h. They were then stimulated with IGF-1 for 24 h in DMEM medium without serum.
During the last 6 h of stimulation, 20 µl of a BrdUrd solution
was added, and the ELISA (20) was performed as suggested by the manufacturer.
DNA Fragmentation Assay--
To determine the frequency of
apoptosis, ES cells were grown for 48 h in 24-well plates, starved
for 24 h, and then stimulated for 24 h. Cells were lysed, DNA
was precipitated, and 10 µg of DNA were separated on a 1.5% agarose gel.
Northern Blotting--
10 µg of total RNA was separated in a
1.5% denaturing agarose gel and transferred to nitrocellulose
membranes by capillary transfer. Northern blots were hybridized with
32P-labeled cDNA probes as previously described (21).
The membranes were washed in 2× saline sodium citrate (SSC), 0.1%
Triton X-100 for 2× 20 min at room temperature, and then in 0.2× SSC,
0.1% Triton X-100 for 2× 10 min at 55 °C. Blots were exposed
overnight with intensifier screens. The generation of cDNA probes
for PI3K regulatory and catalytic isoforms was as described previously (9, 21).
Cell Cycle Analysis--
ES cells were grown to 60%
confluence, washed once with 10 mM phosphate-buffered
saline, serum-starved overnight and then stimulated with IGF-1 for
24 h. Subsequently, the cells were gently trypsinized and fixed in
70% ethanol for 2 h and then washed in phosphate-buffered saline.
DNA was stained by the addition of 0.1% Triton X-100, 200 µg/ml
DNase-free RNase, and 20 µg/ml propidium iodide. Cell cycle detection
was performed on a Becton Dickinson FACS SCAN cell counter at 488-nm
wavelength excitation and 600-nm detection. Data were analyzed with
Cell Quest and ModFitLT V2.0 software.
Adipocyte Differentiation--
ES cells were grown to 80%
confluence, trypsinized, and subsequently grown in normal DMEM with the
addition of LIF in bacterial grade dishes with gentle shaking for 48 to
achieve the formation of aggregates termed embryoid bodies. Embryoid
bodies were then replanted onto cell culture dishes and cultivated in
DMEM without LIF and the addition of 2 nM triiodothyronine
and 85 nM insulin as previously described (19). After 14 days, cells were harvested or stained with oil red for the detection of adipocytes.
Transreporting system for Elk1 and CREB Phosphorylation--
ES
cells were grown for 48 h in normal medium in 6-well plates until
they reached 60-80% confluency. Cells were then washed twice with
phosphate-buffered saline, transfected with luciferase reporter gene
(pFR-Luc), and either Elk1 (pFA-2-Elk1) or CREB (pFA2-CREB)
transactivator domains (all from Stratagene, La Jolla, CA) by
lipid-based transfection (Pfx-6) for 8 h in DMEM without serum.
Subsequently, cells were grown in normal DMEM and then stimulated for
16 h in DMEM containing 1% fetal bovine serum and 10 nM IGF-1. Cells were lysed in luciferase assay buffer
(Stratagene), and luciferase activity was determined (22).
Immunoprecipitation and Immunoblotting--
ES cells were
starved for 12 h and were then equilibrated for another 12 h
in indicated glucose concentrations. One hour prior to stimulation, the
stimulation medium was changed. Cells were lysed after stimulation in
ice-cold lysis buffer (1% Triton X-100, 10% glycerol, 50 mM Hepes pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 5 mM sodium vanadate, 10 µg/ml aprotinin, 5 µg/ml
leupeptin, 1.5 mg/ml benzamidine, and 34 µg/ml phenylmethylsulfonyl
fluoride), sonicated for 15 s, and insoluble material was removed
by centrifugation at 15,000 rpm in a microcentrifuge for 10 min. For
immunoprecipitation, 5 µg of antibodies and 40 µl of a 50% protein
G-agarose slurry were added to 500-1000 µg of proteins for 12 h. The washed immunoprecipitates or 50-100 µg of protein lysate were
separated by 10% SDS-PAGE, Western-transferred on nitrocellulose
membranes, and immunoblotted as previously described (21). Protein
bands were visualized with enhanced chemiluminescence and quantified
using Gelscan 3D software (BioSciTec, Marburg, Germany; Ref.
22).
PI3K Activity--
Immune-complexed PI3K activity was determined
as previously described (21, 22). Immune complexes were incubated in a
55-µl reaction mixture containing 440 µM ATP, 5 µCi
of [ Adenoviral Gene Transfer--
ES cells were grown in normal ES
medium to 60% confluency. Adenovirus at indicated multiplicity of
infection (MOI) (10, 23) was added to the cells in medium without serum
for 1 h, which was exchanged with normal ES medium for 36 h.
The ES cells were then serum-starved for 12 h and subsequently
subjected to various assays.
Phenotype of ES Cells with a Disruption of the Pik3r1 Gene--
ES
cells contain an easily detectable amount of p85
The stoichiometric balance of regulatory and catalytic subunits of PI3K
influences signaling in a given cell type (10, 11). Therefore, we
examined the stoichiometry of regulatory and catalytic isoforms in ES
cells stimulated with IGF-1 by serial immunoprecipitations with
antibodies to p85 Function of Pik3r1 Gene in ES Cells in Signaling to and by
PI3K--
Activation of PI3K by IGF-1 occurs following phosphorylation
of IRS isoforms and association of the PI3K regulatory isoforms with
phosphotyrosines residues. We performed immunoblotting with phosphotyrosine antibodies after immunoprecipitation with antibodies for pY, IRS-1, and IRS-2. No alterations were detected in
phosphorylated IRS isoforms following stimulation with IGF-1 (Fig.
2A). As expected, in ES cells
with a reduction or deletion of p85
To directly examine the effects of a deletion of the Pik3r1
gene upon PI3K activation by IGF-1, lipid kinase activity associated with pY, IRS proteins, and the various regulatory and catalytic subunits of PI3K was determined. Surprisingly, in all cell lines, basal
and IGF-1-stimulated PI3K activity associated with pY, IRS-1, and IRS-2
was not different between wild type, +/ Function of Pik3r1 Gene in Signaling Downstream and Parallel to
PI3K--
Phosphorylated PtdIns generated by PI3K have been shown to
act as second messengers upon a number of downstream signaling molecules. To elucidate whether a disruption in the Pik3r1
gene induced alterations in downstream signaling by PI3K,
phosphorylation of PKB, cdc42, GSK-3, and the transcription factor CREB
was examined. Although p85 Function of Pik3r1 Gene in Proliferation, Apoptosis, and Cell Cycle
Regulation--
Since one of the major functions of PI3K is the
regulation of cellular growth and protection from apoptosis, we
examined proliferation rate, frequency of apoptosis, and regulation of
cell cycle in ES cells with a deletion in the Pik3r1 gene.
The deletion of the p85 Signaling and Cell Cycle Regulation by Adenoviral Re-expression of
p85
The mechanism of alterations in cell cycle of ES cells was examined in
+/+ cells by overexpressing p85 Function of Pik3r1 Gene in Adipocyte Differentiation of ES
Cells--
A role of PI3K has been implicated in the differentiation
of preadipocytes to adipocytes (18, 25). To elucidate whether a
deletion of the Pik3r1 gene resulted in altered adipocyte
differentiation in vitro, ES cell were transformed to
embryoid bodies, which were then cultured in differentiation media
without LIF and the addition of T3 and insulin for 10-14 days. Oil red
staining revealed that all cell lines could be differentiated into
adipocyte-like cells with similar efficiencies of about 20%. The fat
cells were concentrated in the embryoid bodies and scarce in outgrowing
cells (Fig. 7, A and
B) and were functionally active as indicated by expression of the ob gene product leptin (26) (Fig. 7C).
Function of Pik3r1 Gene in Signaling of Differentiated
Adipocyte-like Cells--
To assess whether a deletion of p85 Signaling by PI3K is essential for a wide range of cellular
functions. Recently, it has been shown that the p85 regulatory subunits
of PI3K may act as activators or inhibitors of PI3K. Thus, when
regulatory subunits are expressed at higher stoichiometric levels than
catalytic subunits, the monomeric functionally p85 subunits may inhibit
PI3K activation by competing with functionally active p85/p110
heterodimers for binding sites to phosphorylated IRS proteins (14). In
line with these observations, a reduction of regulatory subunits by
genetic deletion of the Pik3r1 or Pik3r2 genes
can result in increased PI3K signaling and insulin sensitivity in mice
and fibroblast cell lines, since in most insulin-sensitive tissues,
p85 The reduced formation of p85 Unaltered PI3K activation at the level of IRS and p110 catalytic
isoforms in ES cells with a deletion in the Pik3r1 gene did not prevent a reduction in the phosphorylation of PKB, a major downstream target of PI3K. In contrast, GSK-3, which is activated by
PI3K and also independently of PI3K by PKA (27), was equally phosphorylated by IGF-1 in all three cell lines. Whereas these results
indicate a differential dependence of PKB and GSK-3 upon signaling by
p85 Undifferentiated ES cells are rapidly growing cells with an about 50%
S-phase even at starvation. The percentage of cells in S-phase was only
decreased modestly by addition of PI3K inhibitor wortmannin to
wild-type ES cells.2 In line
with these observations, the growth rate of ES cells with a deletion in
the Pik3r1 gene was modestly decreased, which indicates that
the contribution of PI3K to proliferation of ES cell is only minor.
This minor contribution may however change to a major dependence upon
PI3K in embryonic and postnatal development of somatic cells as shown
by embryonic and perinatal lethality of mice with a targeted disruption
of the Pik3ca gene encoding p110 PI3K has been implicated in adipogenic and myogenic differentiation
(34-36). Here, we demonstrated unaltered differentiation of wild-type
and Pik3r1 gene-deleted ES cell lines to adipocytes by
in vitro differentiation. Mice with a homozygous deletion of the Pik3r1 gene may survive for several weeks in an outbred
background and show no morphological alterations implying that the
Pik3r1 gene is not essential for adipocyte differentiation, which is in
line with recent studies showing that brown adipocytes from Pik3r1 gene
knockout animals are viable.3
We also noted in initial experiments that a fraction of ES cells with a
deletion in the Pik3r1 gene differentiated spontaneously in
myocytes depicting synchronized mechanical activity reminiscent of
cardiac muscle cells2 indicating that p85 Re-expression of p85 regulatory subunit of class
IA phosphoinositide 3-kinases (PI3K) is derived
from the Pik3r1 gene, which also yields alternatively spliced variants p50
and p55
. It has been proposed that excess monomeric p85 competes with functional PI3K p85-p110 heterodimers. We examined embryonic stem (ES) cells with heterozygous and homozygous disruptions in the Pik3r gene and found that
wild type ES cells express virtually no monomeric p85
. Although,
IGF-1-stimulated PI3K activity associated with insulin receptor
substrates was unaltered in all cell lines, p85
-null ES cells showed
diminished protein kinase B activation despite increased PI3K activity
associated with the p85
subunit. Furthermore, p85
-null cells
demonstrated growth retardation, increased frequency of apoptosis, and
altered cell cycle regulation with a G0/G1 cell
cycle arrest and up-regulation of p27KIP, whereas signaling
through CREB and MAPK was enhanced. These phenotypes were reversed by
re-expression of p85
via adenoviral gene transfer. Surprisingly, all
ES cell lines could be differentiated into adipocytes. In these
differentiated ES cells, however, compensatory p85
signaling was
lost in p85
-null cells while increased signaling by CREB and MAPK
was still observed. Thus, loss of p85
in ES cells induced
alterations in IGF-1 signaling and regulation of apoptosis and cell
cycle but no defects in differentiation. However, differentiated
ES cells partially lost their ability for compensatory signaling at the
level of PI3K, which may explain some of the defects observed in mice
with homozygous deletion of the Pik3r1 gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain and insulin receptor substrates (IRS) (1-4).
) and Pikr2 (p85
) genes and are
comprised of an NH2-terminal SH3 domain and a BCR homology
region flanked by two proline-rich sites (1-4). In addition, the
Pik3r1 gene yields smaller splicing variants of p55kDa
(p55
; also called AS53) and 50 kDa (p50
) (5-7). Another short
regulatory subunit is p55
; this is structurally similar to p55
but derived from a different gene (8). Both p85
and p85
are
ubiquitously expressed, though p85
appears to predominate in most
tissues. The shorter regulatory isoforms have a more limited expression
indicating cell type-specific roles (5-9).
serves as an
adaptor and activator of heterodimeric PI3K holoenzyme by binding to
phosphorylated receptor-tyrosine kinases and IRS proteins (1-4).
Recently, we have shown that monomeric regulatory subunits not
associated with catalytic subunits may exist and thus act as inhibitors
of PI3K activation (10, 11). Thus, a reduction of the amount of either
p85
or p85
may result in increased activation of PI3K. The
targeted deletion of full-length p85
with preserved expression of
p50
and p55
results in increased insulin sensitivity (12). A
similar phenotype of increased insulin sensitivity is also observed in
mice lacking p85
(13). Likewise, mice with a heterozygous-targeted
deletion of all three isoforms of the Pik3r1 gene p85
,
p55
, and p50
exhibit improved insulin signaling and protection
from the development of diabetes (14, 15). These results suggest an
inhibitory action of PI3K regulatory subunits upon PI3K activation,
which may be explained by formation of p85
monomers, allosteric
inhibition of catalytic isoforms by regulatory subunits, and tyrosine
phosphorylation of p85
at tyrosine 688 (16-18).
as a positive and negative regulator of p85
/p110
heterodimeric PI3K depending upon the relation of regulatory and catalytic isoforms in a given cell type. These results prompted us to
examine signaling by IGF-1 in embryonal stem (ES) cells with a
heterozygous and homozygous deletion in the Pik3r1 gene coding for p85
, p55
, and p50
. We characterized ES cells as a
model of low expression of p85
compared with catalytic subunits resulting in the virtual absence of monomeric regulatory isoforms. Loss
of p85
in ES cells induced increased PI3K activation associated with
p85
, increased apoptosis and alterations in cell cycle, but no
defects in differentiation. However, when ES cells with a loss of
p85
were differentiated, they partially lost their ability for
compensatory signaling at the level of PI3K.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was from Amersham Biosciences. Recombinant
IGF-1 was from Bachem (Bubendorf, Switzerland). 5-Bromodeoxyuridine
(BrdUrd) incorporation ELISAs were from Roche Molecular
Biochemicals. New England Biolabs (Beverly, MA) supplied the
phosphospecific antibodies for serine 473 and threonine 308 of protein
kinase B
/Akt1 (PKB
), serine 133 of cAMP responsive element binder
(CREB), serine 411 of p70S6K, threonine 421/serine 424 of
p70S6K, serine 71 of cdc42, serine 308, threonine 382, threonine 383 of phosphatase and tensin homologue deleted on chromosome
ten (PTEN), serine 795 of retinoblastoma protein (Rb), and serine 21/9
of glycogen synthase kinase 3
/
(GSK-3), GSK-3
/
cross-tide protein along with respective control antibodies for non-phosphorylated kinases, the p27KIP and poly(ADP-ribose) polymerase (PARP)
antibodies, as well as enhanced chemiluminescence reagents. Antibodies
for growth factor bound-2 associated binder-1 (Gab-1), insulin receptor
substrate (IRS) IRS-1, IRS-2, p110
, p110
, monoclonal
phosphotyrosine (pY), and PKB isoforms
,
, and
were from
Upstate Biotechnology (Lake Placid, NY). For the detection of p85
we
applied an antibody raised against the NH2-terminal SH2 of
p85
(Upstate Biotechnology), which recognizes p85
, p55
,
and p50
in immunoblots and exhibits minimal cross-reaction to p85
in immunoprecipitation experiments. Antibodies for phosphorylated
mitogen-activated protein kinases (MAPK; p44/p42) ERK-1, and ERK-2
(pERK Tyr204), control ERK antibodies and
p110pan antibodies were from Santa Cruz Biotechnology. The
monoclonal antibody for p85
(clone T12) was obtained from DPC (Bad
Nauheim, Germany), and the staining kit for
-galactosidase
expression was from Invitrogen (Groningen, Netherlands). Reagents for
SDS-PAGE were from Bio-Rad (Hercules, CA), cell culture reagents were
from Invitrogen (Karlsruhe, Germany), and all other chemicals were from Sigma.
-mercaptoethanol, non-essential amino acids, 1000 units/ml leukemia inhibitory factor (LIF), 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 °C
in a humidified (5% CO2, 95% air) atmosphere. Genotypes
of wild type (+/+), heterozygous knockout (+/
), and homozygous
knockout (
/
) ES cells were verified by PCR with primers for the
wild type and mutated alleles (15).
-32P]ATP, 10 mM MgCl2, and
5 µg of phosphatidylinositol for 20 min at room temperature.
Reactions were stopped by the addition 20 µl of 8 N HCl
and 160 µl of CHCl3/methanol (1:1). The organic phase was
removed by centrifugation and applied to silica gel thin layer
chromatography plates, developed in
CHCl3/CH3OH/H2O/NH4OH (60:47:11.3:2), dried, and visualized by autoradiography. The band
representing phosphatidylinositol 3-phosphate was quantified as
described (21, 22).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
by immunoblotting
in wild type (+/+) cells, and this was reduced by 50% in heterozygous
knockout (+/
) cells and was undetectable in homozygous knockout
(
/
) ES cells (Fig. 1A).
The alternatively spliced isoforms p55
and p50
were not detected
in wild type and the heterozygous or homozygous p85
knockout ES
cells (Fig. 1A). IRS-1, IRS-2, PI3K regulatory subunit
p85
(data not shown), all p110 catalytic isoforms p110
, p110
,
and p110
(p110pan; Fig. 1B), and p110
(data not shown) demonstrated no apparent alterations in expression
levels between the different cell lines as did CREB, MAPKs p42/p44,
PKB, GSK-3, cdc42, and Rb (data not shown). Northern blotting with a
cDNA probe specific for mouse p85
(9) revealed a slight decrease
of p85
transcripts in (
/
) cells (Fig. 1C).
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Fig. 1.
Characterization of Pik3r wild-type (+/+),
heterozygous (+/ ), and homozygous (
/
) knockout stem cells.
A, expression of p85
in stem cells of each genotype.
B, expression of all isoforms of p110 catalytic
subunits demonstrated by a pan-p110 antibody. Cell lysates were
subjected to immunoblotting with indicated specific antibodies.
C, expression of p85
RNA in cells of each genotype.
Equal amounts of total RNA was subjected to Northern blotting and
hybridized with a cDNA probe specific for p85
.
D-F, serial immunodepletion of p85
(D
and E) and p110 (F) using antibodies for p85
and p110. ES cells were starved for 12 h and stimulated with 10 nM IGF-1 for 10 min. Equal amounts of cell lysates were
subjected to three rounds of immunoprecipitation with antibodies for
p85
(D and F) or p110
(E)
followed by immunodetection with p85
(D and E)
and p110
and p110
(p110pan; F).
and p110
/
. A serial immunoprecipitation of
ES cell lysates with antibodies to p85
resulted in the almost complete depletion of p85
by the third immunoprecipitation round in
wild type cells (Fig. 1D), which was accompanied by a
concomitant depletion of phosphorylated IRS isoforms (data not shown).
Similar results were obtained by serial immunoprecipitations using a
mixture of p110
and p110
antibodies depleting completely p85
(Fig. 1E) and phosphorylated IRS proteins (not shown). In
contrast, serial immunoprecipitation of p110 was incomplete in the
third round (Fig. 1F), suggesting that levels of p110
and
p110
exceed the level of p85
, which is low in ES cells with
virtual no expression of monomeric p85
and no residual association
of p85
with IRS proteins.
, binding of phosphorylated IRS
proteins to p85
(Fig. 2B), and also p110
/
catalytic
isoforms was reduced (data not shown). These results indicated
diminished binding of PI3K heterodimer to phosphorylated IRS proteins
in ES cells with a deletion in the Pik3r1 gene. To verify
these results, we examined the binding of p85
to IRS proteins and
p110 catalytic isoforms by immunoprecipitations with specific
antibodies followed by anti-p85
immunoblots (Fig. 2C). In
analogy to the previous experiments, we found that a deletion of p85
resulted in reduced binding to tyrosine-phosphorylated proteins, IRS-1,
IRS-2, p110
, and p110
(Fig. 2C). These results
indicated that phosphorylation of IRS proteins by IGF-1 was unaltered
in the different cell lines. However, a reduction in the amount or
p85
by heterozygous and homozygous deletion of the Pik3r1
gene resulted in reduced binding of regulatory and catalytic subunits
to phosphorylated IRS isoforms.
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Fig. 2.
Effect of disruption of the Pik3r1
gene on IGF-1-induced IRS phosphorylation and IRS-mediated PI3K
activation. A, phosphorylation of IRS-1 and IRS-2.
Cells were starved for 12 h and stimulated with 10 nM
IGF-1 for 10 min. Equal amounts of cell lysates were subjected to
immunoprecipitation with IRS-1 and IRS-2 antibodies followed by
immunodetection with pY antibodies. B, recruitment of
phosphorylated IRS-isoforms to p85 . In A and
B, top panels show representative blots and in
the lower panels, each bar represents the
mean ± S.D. of three independent experiments. They are expressed
assigning a value of 1 to non-stimulated +/+ cells. C,
recruitment of p85
to IRS proteins and catalytic PI3K isoforms.
Cells were treated as in A followed by immunoprecipitation
with antibodies to p85
and immunodetection with indicated
antibodies. Shown are typical blots of n = 3. Abbreviations: +/+, wild-type; +/
, heterozygous p85
knockout;
/
, homozygous p85
knockout.
and
/
cell lines (Fig.
3, A-C) while IGF-1 failed to
stimulate PI3K activity associated with Gab-1 in all cells (Fig.
3D). As expected, PI3K activity associated with p85
was
largely reduced in
/
cells, but a small signal persisted possibly
due to minor cross reaction of the p85
antibody with p85
(Fig.
3E). ES cell with a heterozygous deletion in the
Pik3r1 gene showed only a modest reduction in PI3K activity
associated with p85
. Conversely, PI3K activity associated with the
closely related p85
was up-regulated in +/
and
/
cells (Fig.
3F). There were no alterations in lipid kinase activity
associated with catalytic subunits p110
and p110
(Fig. 3,
G and H). Thus, increased p85
-associated PI3K
activity appears to compensate for the deletion of the p85
at the
level of PI3K.
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Fig. 3.
Effect of disruption of the Pik3r1
gene on IGF-1-stimulated PI3K activity. Cells were starved
for 12 h and stimulated with 10 nM IGF-1 for 10 min.
Equal amounts of cell lysates were subjected to immunoprecipitation
with antibodies to pY (A), IRS-1 (B), IRS-2
(C), Gab-1 (D), p85 (E), p85
(F), p110
(G), and p110
(H).
32P incorporation into phosphatidylinositol 3-phosphate was
quantified after separation by thin layer chromatography using
densitometry. The band shown represents phosphatidylinositol
3-phosphate (PI3P). Top panels show
representative blots and in the lower panels, each
bar represents the mean ± S.D. of 3-5 independent
experiments. They are expressed with assigning a value of 1 to
non-stimulated +/+ cells. Abbreviations: +/+, wild-type; +/
,
heterozygous p85
knockout;
/
, homozygous p85
knockout.
appeared to compensate for deletion of
p85
at the level of PI3-K, phosphorylation of PKB in response to
IGF-1 stimulation in +/
and
/
ES cells was reduced at serine 473 and threonine 308 corresponding to activation of the serine-threonine
kinase (24) compared with wild type ES cells (Fig.
4, A and B),
indicating that p85
was less efficient in stimulating these targets
than p85
. In contrast to the phosphorylation pattern of PKB,
phosphorylation of its downstream target GSK3
/
by IGF-1 was
comparable in all cell types (Fig. 4C). Another target of
PKB is the transcription factor CREB, which is activated by
phosphorylation at serine 133. Interestingly, both basal and IGF-1
stimulated phosphorylation of CREB was increased in +/
and
/
cells (Fig. 4E). A similar pattern of elevated
phosphorylation and activation in knockout cells was also observed at
the level of MAP kinases ERK1 and ERK2 (Fig. 4D). The
increased activation of CREB and MAP kinases was also elucidated with
an increased activation of CREB and Elk-1 in +/
and
/
cells by a
luciferase-based transactivation system (Fig. 4G). These
results indicate a differential dependence of downstream targets of
PI3K by a deletion in the Pik3r1 gene. No difference in
expression (Fig. 4F) and phosphorylation of PTEN at serine
308/threonine 382/threonine 383 (data not shown) was observed in all
three cell types implying that PTEN is not involved in compensatory
signaling in cells with a deletion in the Pik3r1 gene.
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Fig. 4.
Function of Pik3r gene in
signaling downstream and parallel to PI3K. A-F, effect of
disruption of the Pik3r1 gene on PI3K-dependent
and independent signaling and expression of PTEN. Cells were
starved for 12 h and stimulated with 10 nM IGF-1 for
10 min. Equal amounts of cell lysates were subjected to immunoblotting
with antibodies for activated PKB (A and B),
GSK-3 (C), ERKs1/2 (D), CREB (E) as
well as non-activated PTEN (F) and quantified using
densitometry. Top panels show representative blots and in
the lower panels, each bar represents the
mean ± S.D. of three independent experiments. They are expressed
assigning a value of 1 to non-stimulated +/+ cells. Abbreviations: +/+,
wild-type; +/ , heterozygous p85
knockout;
/
, homozygous p85
knockout. G, activation of transcription factors CREB
and Elk-1 by IGF-1 in wild-type and knockout cell lines. ES cell lines
were transfected with CREB and Elk-1 transactivator plasmid as
described under "Experimental Procedures." CREB and Elk-1
phosphorylation were determined by the luciferase activity of a
cotransfected reporter plasmid. ES cells were stimulated with 10 nM IGF-1 for 16 h. Each bar represents the
mean ± S.D. of 4-5 independent experiments.
gene caused a reduction in basal
and IGF-1-stimulated rate of DNA synthesis in p85
+/
and
/
cells (Fig. 5A). Conversely, we observed elevated frequency of apoptosis in p85
+/
und
/
ES
cells, demonstrated by increased DNA laddering (Fig. 5B) and elevated cleavage of PARP (Fig. 5C). Analysis of basal and
IGF-1-stimulated ES cells revealed that these alterations were due to
complex changes in cell cycle regulation in the different cell lines.
Non-stimulated wild type ES cells are rapidly dividing with ~45% in
S-phase. This was increased in +/
cells to 54% and reduced to 39%
in
/
cells. In wild type ES cells, 38% of cells were in
G0/G1 phase, which was reduced to 33% in
p85
+/
and increased to 46% in
/
cells. Thus, ratios of
G0/G1 and S-phases were reversed between p85
+/+ and
/
cells (Fig. 5D), and
/
cells appeared to
be locked in G0/G1 phase. Stimulation of ES
cell lines with IGF-1 prompted a further increase in ratio of S-phase
in all cell lines, which was most pronounced in +/
ES cells and
blunted in
/
ES cells (Fig. 5D). Cell cycle analysis of
sub-G0/G1 cells was applied to estimate the
absolute levels of apoptotic cells in the different cell lines. In a
typical experiment of n = 3, apoptotic cells constituted 3.4% of all counted cells in non-stimulated +/+ cells, 5.12% in +
cells, and 9.5% in
/
cells. IGF-1 stimulation
reduced the percentage of apoptotic cells to 3.17% in +/+ cells, to
3.3% in +/
cells and to 7.06% in
/
cells.
View larger version (48K):
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Fig. 5.
Effect of disruption of the Pik3r1
gene on regulation of proliferation, apoptosis, and cell cycle
regulation. A, proliferation of ES cell lines
stimulated with IGF-1. ES cells were starved overnight and stimulated
for 24 h with 10 nM IGF-1. DNA synthesis was measured
by adding BrdUrd for the last 6 h of the stimulation period and
subsequent detection by ELISA. Each bar represents the
mean ± S.D. of four independent experiments. B,
increased DNA laddering in ES cell lines with a disruption in the
Pik3r1 gene. ES cells were starved for 24 h without
serum and DNA was isolated and separated by an 1% agarose gel. Shown
is a typical result of n = 3. C,
increased cleavage of poly (ADP-ribose) polymerase in ES cells with a
disruption in the gene for Pik3r1 gene. ES cells were
starved overnight, and equal amounts of cell lysates were subjected to
immunoblotting with an antibody recognizing cleaved small (24 kDa) and
large (89 kDa) fragments of PARP. Shown is a representative blot of
n = 3. D, cell cycle regulation in ES
cell lines stimulated with IGF-1. ES cells were starved for 24 h
and subsequently stimulated for 24 h with IGF-1. Cells were gently
trypsinized, fixed in 70% ethanol, stained with propidium iodide and
analyzed by FACS. Shown are representative results of n = 3. Abbreviations: +/+, wild-type; +/ , heterozygous p85
knockout;
/
, homozygous p85
knockout.
in ES Cells with a Deletion in the Pik3r1 Gene--
We applied
adenoviral gene transfer (14, 22) to examine whether alterations in
signaling and cell cycle regulation in ES cells with a deletion of the
Pik3r1 gene could be reversed by re-expression of p85
.
Using an adenovirus harboring the
-galactosidase (lacZ)
gene and galactosidase staining, an MOI between 10 and 200 was
sufficient to infect between 70 and 90% of ES cells. Using an MOI of
20 and 50 of pAdexp85
HA, p85
could be easily overexpressed in
p85
+/+ cells and reconstituted or overexpressed in +/
and
/
cells (Fig. 6A).
An MOI of 20 was sufficient to reconstitute p85
in +/
ES cells at
comparable levels to lacZ infected +/+ ES cells, whereas an MOI of 50 was required in
/
ES cells (Fig. 6A). Overexpression and
reconstitution of p85
in the different cells lines resulted in
increased PKB phosphorylation (Fig. 6B). Similar levels of
PKB phosphorylation was observed in lacZ-transfected +/+ ES
cells compared with +/
and
/
cells infected with low and high
MOIs of pAdexp85
HA, respectively (Fig. 6B).
Interestingly, when an MOI > 50 was used in +/+ and +/
ES
cells, overexpression of p85
inhibited PKB phosphorylation (data not
shown). Re-expression of p85
reversed up-regulation of
cyclin-dependent kinase inhibitor p27KIP in
+/
and
/
ES cells (Fig. 6C) and increased
phosphorylation of Rb (data not shown). Furthermore, we were able to
demonstrate that re-expression of p85
decreased the up-regulated
basal (Fig. 6C) and IGF-1-stimulated CREB phosphorylation in
p85
+/
and
/
cells, and this was paralleled by reduced
phosphorylation of ERKs1/2 (data not shown). Re-expression of p85
also reversed increased PARP cleavage in +/
and
/
cells (Fig.
6D) indicating that increased frequency of apoptosis in +/
and
/
cells was the result of a deletion of the Pik3r1
gene.
View larger version (25K):
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Fig. 6.
Effect of adenoviral-mediated transient
re-expression of p85 in ES cells with a
disruption of the Pik3r1 gene upon signaling and
regulation of apoptosis and cell cycle. ES cells were infected
with low and high multiplicity of infection of adenovirus coding for
HA-tagged p85
(pAdex85
HA). ES cells were infected for 1 h,
grown for additional 36 h and prepared for immunoblotting and cell
cycle analysis as described under "Experimental Procedures."
A, re- and overexpression of p85
in different ES
cell lines demonstrated by immunoblotting for p85
and HA tag of
adenoviral-expressed p85
. B and C,
phosphorylation of PKB in ES cell lines with reconstituted p85
(B). Equal amounts of cell lysates were immunoblotted with
the indicated antibody and quantified using densitometry. Top
panels show representative blots and in the lower
panels, each bar represents the mean ± S.D. of
three independent experiments. They are expressed assigning a value of
1 to stimulated +/+ cells. C, expression levels of
p27KIP and basal phosphorylation of CREB in ES cell lines
with re-expression of p85
. Quantification was performed as in panel
B. D, PARP cleavage in ES cells with
re-expression of p85
. E, cell cycle regulation of ES
cell lines with reconstitution of p85
. ES cells were infected with
high multiplicity of infection of pAdex85
HA and subsequently
stimulated with IGF-1. Cell cycle analysis was performed as described
under "Experimental Procedures." Numbers in brackets
indicated values of non-infected ES cell lines depicted in Fig. 5.
Abbreviations: +/+, wild-type; +/
, heterozygous p85
knockout;
/
, homozygous p85
knockout.
via infection of pAdexp85
HA with
low MOI. In p85
+/
and
/
ES cells, p85
was reconstituted to
similar levels as in native +/+ ES cells p85
by infection with low
and high MOIs of pAdexp85
HA, respectively (Fig. 6E). Here, overexpression of p85
in +/+ ES cells induced a small increase in S-phase with a parallel reduction in G0/G1
and no change in G2/M in non-stimulated cells. No further
increase in percentage of cells in S-phase was observed in +/+ ES cells
stimulated with IGF-1. In p85
+/
cells, reconstitution of p85
caused no alterations in non-stimulated cells, whereas the percentage
of ES cells in S-phase following IGF-1 treatment was reduced by
re-expression of p85
. Reconstitution of p85
in
/
cells
increased the percentage of cells in S-phase by 10% and reduced
G0/G1 phase to similar levels as in
non-infected +/+ cells (Figs. 5D and 6E) in
non-stimulated ES cells. A similar reversal of cell cycle alterations
by re-expression of p85
was also observed in
/
cells stimulated
with IGF-1, which displayed a similar cell cycle as native +/+ cells
stimulated with IGF-1 mainly by reduction of
G0/G1-phase and elevation of S-phase (Fig.
6E). Analysis of sub-G0/G1 cells was
used to assess alterations of apoptotic cells by over- and
re-expression of p85
in the different non-stimulated cell lines
compared with non-transfected cells. In a typical experiment of
n = 3, overexpression of p85
in +/+ cells reduced
the number of apoptotic cells from 3.17 to 2.28%. Reconstitution of
p85
in +/
cells reduced the number of apoptotic cells from 5.12 to
3.64% and from 9.5 to 4.76% in
/
cells.
View larger version (60K):
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Fig. 7.
Effect of disruption of the Pik3r1
gene on differentiation of ES cells into adipocytes. ES
cells were differentiated into embryoid bodies and treated with normal
medium without LIF (A) or a differentiation medium
(B) containing T3 and insulin for 14 days. Cells were fixed
and stained with oil red. Lower panels depict stained cells
at higher magnification. C, detection of leptin
in differentiated and undifferentiated ES cells. Equal amounts of cell
lysates were subjected to immunoblotting with monoclonal leptin
antibody. Shown is a representative blot. Abbreviations: +/+,
wild-type; +/ , heterozygous p85
knockout;
/
, homozygous p85
knockout.
induced similar defects in signaling in differentiated adipocyte-like
cells compared with undifferentiated ES cells, PI3K activation by IGF-1
was examined at the level of IRS proteins and regulatory and catalytic
subunits of PI3K (Fig. 8,
A-G). In contrast to the unaltered signaling in
undifferentiated ES cells (Fig. 3, A-C), PI3K activity
association with phosphorylated signaling proteins (Fig.
8A), IRS-1 (Fig. 8B), and IRS-2 (Fig.
8C) was reduced in
/
ES cells, but not in +/
ES cells.
These alterations were accompanied by reduced PI3K activity associated
with p85
(Fig. 8D) in +/
and
/
ES cells similar to
signaling by p85
in undifferentiated ES cells (Fig. 3E).
However, in differentiated adipocyte-like ES cells, PI3K activity
association with p85
(Fig. 8E) was not increased in +/
and
/
ES cells as it was in undifferentiated ES cells (Fig.
3F). Failure of compensatory signaling by p85
resulted in
reduced PI3K activity associated with catalytic subunits p110
and
p110
(Fig. 8, F and G). Interestingly,
downstream signaling of PI3K was similar in differentiated and
undifferentiated ES cells. Phosphorylation of PKB was reduced and
phosphorylation of ERK1/2 and CREB by IGF-1 was increased in +/
and
/
ES cells (Fig. 8, H-K).
View larger version (29K):
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Fig. 8.
Effect of disruption of the Pik3r1
gene on PI3K-dependent and -independent signaling in
ES cells differentiated into adipocytes. Cells were starved for
12 h and stimulated with 10 nM IGF-1 for 10 min. Equal
amounts of cell lysates were subjected to immunoprecipitation with
antibodies to pY (A), IRS-1 (B), IRS-2
(C), p85 (D), p85
(E), p110
(F), and p110
(G). PI3K assays were performed
as described under "Experimental Procedures." The band shown
represents phosphatidylinositol 3-phosphate (PI3P).
I-K, phosphorylation of PKB (H), ERK1/2
(I), and CREB (J) by IGF-1 in differentiated ES
cells. Equal amounts of cell lysates were subjected to immunoblotting
with indicated antibodies. Top panels show representative
blots and in the lower panels, each bar
represents the mean ± S.D. of 3-5 independent experiments. They
are expressed assigning a value of 1 to non-stimulated +/+ cells.
Abbreviations: +/+, wild-type; +/
, heterozygous p85
knockout;
/
, homozygous p85
knockout.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is stoichiometrically overexpressed compared with p110 catalytic
subunits (9, 10-14). Here, we demonstrated that ES cells are a unique
cellular model in which p85
is not overexpressed compared with p110
catalytic subunits. This stoichiometric imbalance prevents increased
PI3K activation in ES cells with a heterozygous depletion of the
Pik3r1 gene, because generation of functionally active
p85
/p110 heterodimers is limited by the expression of p85
. Thus,
interaction of p85
and p110 is almost saturated in ES cells and
reduced in heterozygous and homozygous Pik3r1 gene knockout
ES cells according to the deletion of the p85
gene. Furthermore,
this phenotype prevents the binding of monomeric p85
to
phosphorylated IRS-isoforms in IGF-1 stimulated ES cells as
demonstrated by similar depletion kinetics using antibodies for p85
and catalytic p110 subunits. The special stoichiometry of excess p110
compared with p85
may also explain why we do not observe a marked
reduction in p110 levels by the reduction of regulatory isoforms as
described in other cell types with an excess of p85
(12). Since it
not likely that free p110 monomers exist due to thermal instability
(16), p85
may serve to stabilize p110 in ES cells.
/p110 heterodimers in heterozygous and
homozygous Pik3r1 gene knockout cells explains decreased binding to
phosphorylated IRS proteins following IGF-1 stimulation. Surprisingly,
PI3K activity associated with IRS-1 and IRS-2 is not decreased in ES
cells with a heterozygous or homozygous deletion in the
Pik3r1 gene. Likewise, PI3K activity associated with p110
and p110
is approximately equal in all three cell lines. This experimental paradox may be partially explained by increased
recruitment of p85
in cells with a reduction in p85
protein
levels. Binding of phosphorylated IRS proteins to catalytic p110
subunits was decreased in +/
and
/
cells concomitantly to the
reduction of p85
possibly reflecting the less efficient binding of
p85
to IRS proteins. Interestingly, when ES cells were
differentiated to adipocytes, PI3K activity associated with IRS and
p110 proteins was reduced in homozygous Pik3r1 gene knockout
cells. This phenomenon was associated with a decrease of
p85
-associated PI3K activity in ES cells with a deletion in p85
.
These results indicate that undifferentiated ES cells are able to
compensate decreased p85
signaling by increase in PI3K activity of
p85
, which is not preserved in differentiated ES cells.
-associated PI3K, we demonstrated that a reduction or loss of
p85
induced up-regulation in the stimulation of the Ras-Raf-MAPK and
the cAMP-dependent protein kinase (PKA)/CREB pathway. Thus,
undifferentiated ES cells possess an enormous flexibility to compensate
for the reduction of signaling by p85
, which is partially lost by
differentiation. We were able to exclude alteration in the expression
levels of PIP3 phosphatase PTEN (28, 29) as a possible mechanism of
compensation for the loss of p85
.
and the
Pik3r1 gene encoding p85
, p55
and p50
, respectively (13, 30). We demonstrated reduced percentage of cells in S-phase and an
increase in G0/G1 phase in p85
/
cells
under basal conditions and after stimulation with IGF-1 indicating that
a deletion of the Pik3r1 gene induced a partial
G0/G1 block, possibly by up-regulation of
p27KIP1, a major inhibitor of G1
cyclin-dependent kinases (28, 31). Increased levels of
p27KIP1 in Pik3r1 gene knockout cells may be
explained by direct transcriptional regulation of the
p27KIP1 gene by forkhead transcription factors, which are
negatively regulated by the PI3K/PKB pathway (32). Unexpectedly, we
demonstrated increased percentage of cells in S-phase in
non-stimulated and IGF-1-stimulated ES cells with a partial reduction
of p85
by a heterozygous deletion of the Pi3kr1 gene.
Under normal conditions, an increased percentage of cells in S-phase
should lead to increased BrdUrd incorporation. This, however was not
observed in +/
ES cells possibly to increased frequency of apoptosis,
which may reverse increased proliferation of cells by premature cell
death. Increased frequency of apoptosis in +/
and
/
ES cells
correlated with decreased PKB activation. Conversely, PKB
has been
shown to be the major effector of a model of increased PI3K activity, the PTEN knockout ES cells (29). Thus, decreased PKB activation in
Pik3r1 gene knockout ES cells may affect regulation of
apoptosis by altering activation of PKB effectors such as BAD and FKHR
(29, 33).
is not
essential for myogenic differentiation.
by adenoviral gene transfer was used to examine
whether the phenotype of ES cells with a heterozygous and homozygous
deletion in the Pik3r1 gene was caused by clonal variation
or by the reduction or loss of p85
itself. Since we demonstrated
that re-expression of p85
at similar levels as in wild-type cells
was able to reverse the phenotype of +/
and
/
ES cells, clonal
selection appears unlikely. Interestingly, we noted in pilot
experiments that p85
could be easily overexpressed in all cell lines
by adenoviral gene transfer using high MOIs between 100 and 200. When
p85
was overexpressed in ES cells of all genotypes, it acted as a
negative regulator of PKB activity in analogy to experiments performed
in fibroblasts and L6 myotubes (12, 14). Thus, ES cells are an ideal
cellular model to study the balance of different combinations of
regulatory and catalytic subunits, which may provide further
information upon the cellular complexity and physiology of signaling by
class 1A PI3K.
![]() |
ACKNOWLEDGEMENT |
---|
We thank H. Schmidt for technical support.
![]() |
FOOTNOTES |
---|
*
This research was supported by Grant Ho 1762/21 from the
Deutsche Forschungsgemeinschaft (to D. H.) and National Institutes of Health Grant GM41890 (to L. C. C.). Parts of this study are contained in the medical thesis of D. Hallmann.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.
§ These authors contributed equally.
To whom correspondence should be addressed: Dept. of Internal
Medicine, Division of Gastroenterology and Metabolism,
Philipps-University, Baldingerstrasse, D-35033 Marburg, Germany. Tel.:
49-6421-2862780; Fax: 49-6421-2868922; E-mail:
hoerschd@post.med.unimarburg.de.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M208451200
2 D. Hallmann and D. Hörsch, unpublished observations.
3 K. Ueki, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PI3K, phosphoinositide 3-kinase; BrdUrd, 5-bromodeoxyuridine; CREB, cAMP responsive element-binding protein; ES, embryonic stem; Gab-1, growth factor bound-2-associated binder-1; GSK-3, glycogen synthase kinase 3; IGF-1, insulin-like growth factor-1; IRS, insulin receptor substrate; LIF, leukemia inhibitory factor; MAPK, mitogen-activated protein kinase; MOI, multiplicity of infection; PARP, poly(ADP-ribose) polymerase; PI, phosphoinositides; PKB, protein kinase B/Akt; PtdIns, phosphatidylinositol; PTEN, phosphatase and tensin homologue deleted on chromosome ten; pY, phosphotyrosine; Rb, retinoblastoma protein; SSC, saline sodium citrate; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay.
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