(Investigative Ophthalmology and Visual Science. 2000;41:844-849.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Phosphatidylinositol 3-Kinase in Bovine Lens and Its Stimulation by Insulin and IGF-1
Gudiseva Chandrasekher1 and
Haydee E. P. Bazan1
1 From the Louisiana State University Medical Center Eye Center and Neuroscience Center, New Orleans, Louisiana.
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Abstract
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PURPOSE. To identify and characterize phosphatidylinositol 3-kinase (PI-3K) in
the lens and to study its involvement as a signal mediator in lens
epithelial cells exposed to insulin and insulin-like growth factor
(IGF)-1, which are known to induce lens epithelial cell proliferation
and differentiation into fiber cells.
METHODS. Concentric fiber cell layers from single bovine lens were prepared by
dissolution in buffer. PI-3K activity in capsule-epithelium and fiber
cell layers was determined after immunoprecipitation with antibodies
against p85, the regulatory subunit of PI-3K. High-performance liquid
chromatography on an ion exchange column (Partisil-SAX; Whatman,
Maidstone, United Kingdom) was used to identify PI-3K reaction
products. Cultured bovine lens epithelial cells were stimulated with
insulin or IGF-1, and PI-3K activity was determined after
immunoprecipitation with antibody against phosphotyrosine. Association
of p85 with other proteins after stimulation was determined in anti-p85
immunoprecipitates by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE) and Western immunoblot analysis using
anti-phosphotyrosine antibody.
RESULTS. PI-3K activity was found in both lens epithelial cells and fiber
cells. The highest specific activity was found in the
capsule-epithelium, but there was considerable activity in other fiber
cell layers. Insulin and IGF-1 stimulated the PI-3K activity in
epithelial cells in culture by more than 100%, and activation of the
enzyme resulted in tyrosine phosphorylation of the p85 subunit. After
stimulation, the p85 subunit of PI-3K was associated with 100- and
180-kDa tyrosine phosphorylated proteins.
CONCLUSIONS. The activation of PI-3K and its association with specific
tyrosine-phosphorylated proteins may be important in insulin and IGF-1
signal transduction in lens epithelial cells. The presence of
significant PI-3K activity throughout the lens further suggests that
this signal transduction enzyme is sustained in fiber
cells.
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Introduction
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Lens tissue is formed from a monolayer of cuboidal
epithelial cells at the anterior surface that proliferate, elongate,
and eventually differentiate and become fiber cells. These fiber cells
constitute the main body of the lens. Insulin and insulin-like growth
factor (IGF)-1 have been shown to induce lens epithelial cell
proliferation in chicken, bovine, and rabbit lens and differentiation
in rat and chicken lens (reviewed in Reference 1). Other growth
factors, such as fibroblast growth factor and platelet-derived growth
factor, have also been shown to affect proliferation and
differentiation processes in rat and mice lens.1
2
Growth
factors bind to their tyrosine kinase receptors and generate signals in
the cells that are transduced by several intracellular signal
mediators. The actual molecules that participate in the growth
factor–mediated signal transduction in lens cells are not known at
this point.
PI-3K is an important component in the signal transduction cascade
initiated by various receptor tyrosine kinase specific growth factors.
This kinase is a heterodimeric protein with a 110-kDa catalytic subunit
(p110) and an 85-kDa regulatory-adaptor subunit (p85) with two Src
homology (SH2) domains,3
that interact with the cytosolic
portion of the tyrosine kinase domain of the receptor. PI-3K
phosphorylates phosphatidylinositol lipids at the 3' position of the
inositol ring, generating an important group of second messengers,
namely, phosphatidylinositol 3-phosphate (PI-3P), phosphatidylinositol
3,4-bis phosphate (PI-3,4P2), and
phosphatidylinositol 3,4,5-triphosphate
(PI-3,4,5P3).3
Several studies have
implicated PI-3K through its products in the regulation of cell
proliferation, cellular differentiation, cytoskeletal reorganization,
membrane trafficking, prevention of apoptosis, glucose transport,
survival and metabolism (reviewed in Reference 4). The present study
shows for the first time that insulin and IGF-1 stimulate PI-3K in
bovine lens epithelial cells, suggesting its role as a mediator in
growth factor–dependent cellular processes such as proliferation and
differentiation. We also show that PI-3K is present throughout the lens
and that fiber cells retain this enzyme after their differentiation.
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Materials and Methods
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Materials
Polyclonal and monoclonal antibodies for the p85 subunit of PI-3K
and anti-phosphotyrosine, (anti-PY) and IGF-1 were from Upstate
Biotechnology, (Lake Placid, NY). All electrophoresis reagents were
from Bio-Rad, (Richmond, CA). [
33P]ATP,
phosphatidyl [2-3H] inositol-4-mono phosphate
(3H-PI-4P), and phosphatidyl
[2-3H] inositol 4,5 bis phosphate
(3H-PI-4,5P2) were from
Amersham (Arlington Heights, IL). Insulin, Wortmannin,
Dulbecco’s modified Eagle’s medium (DMEM), phosphatidylinositol (PI),
PI-4P and PI-4,5P2, and antibiotic-antimycotic solutions
were from Sigma (St. Louis, MO).
Epithelial Cell Culture
Lens from calf eyes (Pel-Freeze Biologicals, Rogers, AR), were
separated, the capsule with adhering epithelial cells was dissected
from the lens, and primary cultures of epithelial cells were cultured,
as reported5
in DMEM supplemented with 10% fetal calf
serum (Gibco, Grand Island, NY) and 1% antibiotic-antimycotic solution
at 37°C in a 5% CO2-95%
O2. Second-passage cells were used in all
experiments.
Preparation of Cultured Epithelial and Lens Fiber Cell Layer
Extracts
Concentric fiber cell layers from a single decapsulated bovine
lens (weighing approximately 2 g) were obtained by progressively
dissolving the lens6
with gentle and constant agitation in
3 to 5 ml lysis buffer (20 mM HEPES, 2 mM Mg 2+,
2 mM EGTA, 2 mM orthovanadate, 2 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 0.1 mM leupeptin, and 0.25 M
sucrose, 1% Triton X-100 and 0.5% NP-40). Six layers were prepared
from one lens. Layers were numbered consecutively from the periphery to
the center of the lens: layer 1 represented the outermost fiber cells
of the lens and layer 6 was designated as the nucleus. The capsule with
adherent epithelial cells was also collected. All lens cell fractions
and stimulated cultured epithelial cells were homogenized in lysis
buffer using a glass homogenizer and centrifuged at
100,000g for 30 minutes, and the supernatant was used as
the source of PI-3K. All the operations were performed at
4oC. Protein content of the extracts was determined by
the method of Lowry et al.7
Immunoprecipitation and Western Blot Analysis
Cultured epithelial cell extracts (0.3–0.6 mg protein) or fiber
cell extracts (1–10 mg protein) were immunoprecipitated with 3 to 5
µl of anti-p85 or 3 to 5 µg of anti-PY polyclonal antibodies, as
described before.7
Proteins present in immunoprecipitates
were either assayed for PI-3K activity or subjected to sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 9% gel),
followed by western transfer to nitrocellulose membranes.8
The transferred proteins were probed with anti-p85 subunit or anti-PY
antibodies. The protein bands were visualized using an enhanced
chemiluminescence kit (Amersham). Negative controls without the primary
antibody produced no bands. The chemiluminescence bands on x-ray film
were quantified as described earlier.8
PI-3K Assay
PI-3K activity was determined essentially as described
previously,8
using PI as substrate (37.5 µg). In some
experiments, PI-4P and PI-4,5P2 were also used as
substrates (37.5 µg each). The PI-3K reaction products were separated
on thin-layer chromatography (TLC) plates (Merck, Darmstadt, Germany)
and scanned for 33P-labeled 3' PI spots using an
image analyzer (Packard, Meriden, CT).8
In some cases,
radiolabeled spots on plates were scraped and lipids extracted for
further identification by high-performance liquid chromatography
(HPLC).
HPLC Analysis of PI-3K Reaction Products
Products of PI-3K were deacylated8
and the
glyceroinositol-phosphates separated on an anion exchange HPLC column
(Partisil-SAX)9
with a gradient from 0 to 1.0 M
(NH4)2HPO4
(pH 3.8) in water for 120 minutes (pump A, water; pump B,
(NH4)2HPO4;
0% B for 10 minutes to 25% B with a gradient over 60 minutes, to 50%
B over 30 minutes, and 100% B for the last 20 minutes). The compounds
were quantified with a radiomatic detector connected on-line.
Deacylated 3H-PI-4P and
3H-PI-4,5P2 were used as
authentic standards, because 3H-PI-3P and
3H-PI-3,4P2 compounds are
not commercially available. The identification was based on the fact
that the isomers of PI-3P and PI-4P and of
PI-3,4P2 and PI-4,5P2 have
different retention times.8
Statistical significance of the data was determined using Student’s
t-test.
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Results
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PI-3K in Bovine Lens Epithelial and Fiber Cells
Initial experiments were conducted to characterize PI-3K
activity in lens fiber cells after immunoprecipitating the enzyme with
anti-p85 antibody that immunoprecipitates the 85-kDa regulatory subunit
(p85) and the associated p110, the catalytic subunit of PI-3K. The
enzyme converted the substrate PI to PI-3P (Fig. 1A
, lane 3); it also less efficiently phosphorylated PI-4P and
PI-4,5P2 to PI-3,4P2 and
PI-3,4,5P3, respectively (Fig. 1A
, lanes 1and 2).
Wortmannin, a selective inhibitor of PI-3K that covalently binds to the
catalytic p110 subunit inhibited the enzyme activity (Fig. 1A
, line 4).
The three PI-3K products separated by TLC were further identified by
HPLC analysis (Fig. 1B)
and their retention times were compared with
deacylated standards of 3H-PI-4P and
3H-PI-4,5P2.9
The characterized PI-3K activity was distributed throughout the lens
(Fig. 1C)
. The activity, expressed per milligram lens protein, was
higher in the capsule-epithelial layer than in the remainder of the
lens; but significant amounts of the activity were present in fiber
cell layers. The lower activity in the inner layers was due to dilution
of the enzyme protein by massive amounts of crystallins, as can be seen
when total activity per layer was calculated. The p85 protein in the
immunoprecipitates of the lens layers was also identified after Western
blot, with anti-p85 antibody. Similar to enzyme activity, a gradient of
p85 protein was observed in lens with the highest concentration in the
capsule-epithelial layer (Fig. 1D)
.
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Figure 1. Characterization of PI-3K in bovine lens. (A)
Synthesis of different 3' PIs by PI-3K. The PI-3K activity in anti-p85
immunoprecipitates from fiber cells extracts was assayed using
PI-4,5P2 (lane 1), PI-4P (lane
2), and PI (lanes 3 and 4) as
substrates and the products separated by TLC. Twenty nM Wortmannin (Wt,
lane 4), a specific inhibitor of PI-3K, inhibited the
formation of PI-3P. (B) Separation and identification of
PI-3K products from crude lens fiber cells extracts by HPLC. The bands
were extracted from a TLC plate, and the deacylated products, gPI-3P
(tracing 2), gPI-3,4P2 (tracing
4), and gPI-3,4,5 PI3 (tracing 5)
derived from PI-3P, PI-3,4P2, and PI-3,4,5P3
were run and compared with deacylated standards. A mixture of gPI-3P
and authentic standard 3H-gPI-4P is shown in tracing
1. Tracing 3 represents the elution profile of a
mixture of gPI-3,4P2 and authentic standard 3H
gPI-4,5P2. (C) Protein fractions from different
lens regions were prepared as described in the Methods section. PI-3K
activity was assayed using PI as substrate. PI-3P bands on TLC plates
were quantified by image analysis and expressed as counts per million
per milligram protein. The values ± SD correspond to two
different experiments. The average total amount of activity in each
layer was indicated above the corresponding bar. (D)
Identification of p85, the regulatory subunit of PI-3K in different
lens layers. Cell extracts (1 mg protein) of each layer were
immunoprecipitated with a polyclonal anti-p85 antibody, subjected to
SDS-PAGE, and identified by Western blot analysis with monoclonal
anti-p85 antibody (the amount of protein used for immunoprecipitation
in layer 5 [*] and nucleus [**] was four and eight times
higher than in the other fractions). The experiment was repeated once
with similar results. The density units obtained for the p85 band of
capsule-epithelium per milligram protein, were normalized to 1 and the
total amount of p85 in each layer was calculated, relative to the
amount present in capsule-epithelium. The total units thus calculated
for p85 in each layer are: capsule-epithelium (2.0 units), layer 1
(19.4), layer 2 (19.2), layer 3 (34.6), layer 4 (33.0), layer 5 (11.4),
and nucleus (4.2).
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Insulin and IGF-1 Stimulate PI-3K Activity in Lens Epithelial Cells
To understand the relevance of PI-3K, we investigated whether, in
lens epithelial cells, the growth factors insulin and IGF-1 can
modulate its activity. For these studies, bovine lens epithelial cells
in culture were incubated with insulin or IGF-1 and the proteins
immunoprecipitated with anti-PY antibody to determine whether the PI-3K
activity associates with the tyrosine phosphorylated
proteins.10
Both growth factors caused more than a 100%
increase in PI-3K activity immunoprecipitated by anti-PY antibody after
10 and 15 minutes of stimulation and reached near basal levels at 45
minutes (Fig. 2)
. To examine the presence of p85 in anti-PY immunoprecipitates, cells
were exposed for 10 and 45 minutes to insulin or IGF-1, the cell
extracts immunoprecipitated with anti-PY antibody, and the proteins
analyzed by Western blot with anti-p85 antibody. A single band with a
molecular weight of 85 kDa was found (Fig. 3A ). Stimulation of the cells for 10 minutes caused more than a 200%
increase in the intensity of the band (Fig. 3B)
demonstrating a
correlation between PI-3K activity and an increase in the p85 in the
anti-PY immunoprecipitates.
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Figure 2. PI-3K activation after insulin and IGF-1 stimulation in bovine lens
epithelial cells. Epithelial cells in culture were incubated overnight
in serum-free medium and then treated with insulin (100 nM) or IGF-1
(50 nM). Cell lysates containing the same amount of protein were
subjected to immunoprecipitation with a polyclonal anti-PY antibody.
Immunoprecipitates were then assayed for PI-3K activity, the enzyme
product PI-3P was separated by TLC, and the radiolabeled PI-3P band
quantified with an image analyzer. Because different experiments were
performed with insulin and IGF-1, control (without growth factors) bars
for each condition are shown separately. Values are expressed as counts
per million per milligram protein and are the average of three
experiments ± SD. *, Significant difference
(P < 0.05) compared with unstimulated cells.
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Figure 3. Presence of the p85 subunit of PI-3K in anti-PY immunoprecipitates
after insulin and IGF-1 stimulation. (A) Cultured epithelial
cells were incubated overnight without serum and then incubated with
insulin (100 nM) or IGF-1 (50 nM). Proteins were immunoprecipitated
with a polyclonal anti-PY antibody, separated by SDS-PAGE, transferred
to nitrocellulose membrane, and probed with a monoclonal anti-p85
antibody. (B) Quantification of the p85 protein band by
densitometric scanning. The experiment was repeated once with similar
results.
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Association of the p85 Subunit of PI-3K with Other
Tyrosine-Phosphorylated Proteins in Stimulated Lens Epithelial Cells
To determine whether insulin and IGF-1 signaling leads
to the interaction of p85 with other tyrosine phosphorylated proteins,
bovine lens epithelial cells in culture were stimulated with IGF-1, and
proteins were immunoprecipitated with anti-p85 antibody and subjected
to SDS-PAGE followed by Western blot using anti-PY antibody. In
addition to the p85 subunit there were 100- and 180-kDa bands (Fig. 4A
) indicating that these proteins coimmunoprecipitate with the p85. The
level of protein phosphorylation after IGF stimulation increased
between 5 and 15 minutes. Blots probed with anti-p85 antibody showed
the same amount of p85 protein in all samples (Fig. 4B)
.
Insulin-stimulated lens epithelial cells showed a similar association
of p85 with 100- and 180-kDa proteins (data not shown).
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Figure 4. Coimmunoprecipitation of tyrosine-phosphorylated proteins with p85
after IGF-1 stimulation. Lens epithelial cells in culture were
incubated with IGF-1 (50 nM) for up to 45 minutes. Cell lysates were
immunoprecipitated with a polyclonal anti-p85 antibody, resolved by
SDS-PAGE, and analyzed by Western immunoblot analysis with monoclonal
anti-PY (A) or anti-p85 antibodies (B). This
experiment was repeated once with similar results. Lane
C, controls (without IGF-1).
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Discussion
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Growth factors are important for cellular processes such as
proliferation, elongation, and differentiation that occur during the
development and growth of ocular lens. The precise pathways that
transduce the growth factor signals in lens have not been previously
studied. In the current study, we show that in lens epithelial cells,
insulin and IGF-1 stimulate PI-3K activity, which can lead to the
generation of 3' PIs, which have been shown to trigger a cascade of
signal transduction reactions involved in mitogenic and differentiation
processes.3
4
There was more than a 100% increase in
PI-3K activity over controls after insulin and IGF-1 stimulation.
Similar increases have also been observed in other cell types when
stimulated with agonists. Previous studies have shown increased
turnover of PI during epithelial cell division and a rapid decline
during their differentiation,11
and it was suggested that
phospholipase C–dependent PI turnover pathway may be involved.
However, this pathway was not found to be influenced by
insulin.12
PI and other PIs when phosphorylated by PI-3K
are not substrates for phospholipase C but are components of an
intracellular signaling system important in the control of cellular
functions such as growth and survival.3
4
We also show the
tyrosine phosphorylation of the p85 after insulin and IGF-1
stimulation. There was a correlation between phosphorylation of the p85
and activation of PI-3K, suggesting that phosphorylation may be
important in the activation of the enzyme in lens epithelial cells.
Tyrosine phosphorylation of the p85 subunit has been shown after growth
factor activation in other cell types,13
14
although it is
not clear whether it is necessary for enzyme activation.
After insulin and IGF-1 stimulation, p85 is associated with other
tyrosine-phosphorylated proteins. We speculate that the 100-kDa protein
could be the insulin-IGF receptor ß subunit (95–105 kDa) with
intrinsic tyrosine kinase activity and the 180-kDa protein, the IRS-1
(165–185 kDa), because these proteins have been shown to be
phosphorylated and to form a signaling complex with
PI-3K,10
15
facilitating its translocation to the plasma
membrane (preliminary experiments to identify IRS-1 with two
commercially available anti-IRS-1 antibodies were unsuccessful,
probably because of differences in species specificity of the
antibodies). Several tyrosine residues of IRS-1 are phosphorylated
after growth factor stimulation.16
In our experiments we
have observed a broad, not well-separated 180-kDa (Fig. 4B)
band that
could be a result of differential phosphorylation of tyrosine residues
on IRS-1 molecules.
We also show that fiber cells retained PI-3K activity after
differentiation. The metabolic activity of epithelial cells is high,
because these cells proliferate, elongate, and, finally, differentiate
into fiber cells to keep the lens growing. In addition, fiber cells
depend on this epithelial cell layer for the maintenance of
transparency. This could be the reason for high activity of PI-3K in
epithelial cells, and this enzyme may play a role in these processes.
However, it is interesting to note that PI-3K activity is not confined
only to the epithelium and elongating and differentiating superficial
fiber cells and that a considerable activity is also present in inner
fiber cell layers (it should be noted that during lens dissolution the
amount of fiber cell protein recovered in the inner layers 3 and 4 was
higher than in other layers). Taking into account the enzyme activity
and the amount of p85 found in each fraction (Fig. 1)
, we calculated
the theoretical specific activity of the enzyme in epithelial cells as
3750 cpm/unit p85. This activity decreased to approximately 1500
cpm/unit p85 in superficial fiber cell layer (layer 1) and was
maintained at similar values up to the nucleus, indicating that this
protein did not undergo significant modifications in the fiber cells
that could result in loss of catalytic activity. The relevance of PI-3K
presence in fully matured and even in aged fiber cells is not clear at
present. It can be argued that the enzyme is present as a result of the
absence of active turnover of proteins in lens. However, several
enzymes have also been shown to be active and participate in many
reactions that have relevance to lens function. In chicken lens,
insulin and IGF-1 receptors have been shown to decrease but not
completely disappear.17
18
Therefore, it is possible that
PI-3K–mediated signal transduction is sustained in fiber cells and
that this enzyme may serve as a survival factor from its role in a wide
array of functions.
In conclusion, we have shown that in lens epithelial cells, insulin and
IGF-1 induce tyrosine phosphorylation of the p85 subunit, as well as
activation of PI-3K. Insulin and IGF-1 have been shown to augment
fibroblast growth factor–induced fiber cell
differentiation.19
20
The activation of PI-3K and its
binding to specific tyrosine-phosphorylated proteins may be an
important signaling mechanism in the regulation of insulin and IGF-1
actions in lens, such as proliferation of epithelial cells and their
differentiation into fiber cells. The presence of PI-3K throughout the
lens implicates sustained PI-3K signal transduction in fully
differentiated fiber cells. The role of this enzyme during
differentiation induced by growth factors and its function in
maintaining the normal metabolism of fiber cells is currently under
investigation.
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Footnotes
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Supported by National Institutes of Health Grant EY-06635 and Core Grant EY-02377.
Submitted for publication July 15, 1999; revised October 1, 1999; accepted October 26, 1999.
Commercial relationships policy: N.
Presented in part at the annual meetings of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, 1998 and 1999.
Corresponding author: Haydee E. P. Bazan, LSUMC Eye Center and Neuroscience Center, 2020 Gravier Street, Suite D, New Orleans, LA 70112. hbazan1{at}lsumc.edu
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Abstract
Introduction
