Secretion of Annexin II via Activation of Insulin Receptor and
Insulin-like Growth Factor Receptor*
Wei-Qin
Zhao
§¶,
Gina H.
Chen
,
Hui
Chen
,
Alessia
Pascale
**,
Lakshmi
Ravindranath
§,
Michael J.
Quon
, and
Daniel L.
Alkon
§
From the
Laboratory of Adaptive Systems, NINDS,
National Institutes of Health, Bethesda, Maryland 20892, § Blanchette Rockefeller Neurosciences Institutes,
Rockville, Maryland 20850,
Diabetes Unit, LCI, NCCAM, National
Institutes of Health, Bethesda, Maryland 20892, and the
** Department of Experimental and Applied Pharmacology,
University of Pavia, Pavia, Italy
Received for publication, October 15, 2002, and in revised form, November 8, 2002
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ABSTRACT |
Annexin II is secreted into the extracellular
environment, where, via interactions with specific proteases and
extracellular matrix proteins, it participates in plasminogen
activation, cell adhesion, and tumor metastasis and invasion. However,
mechanisms regulating annexin II transport across the cellular membrane
are unknown. In this study, we used coimmunoprecipitation to show that
Annexin-II was bound to insulin and insulin-like growth factor-1 (IGF-1) receptors in PC12 cells and NIH-3T3 cells overexpressing insulin (NIH-3T3IR) or IGF-1 receptor
(NIH-3T3IGF-1R). Stimulation of insulin and IGF-1 receptors
by insulin caused a temporary dissociation of annexin II from these
receptors, which was accompanied by an increased amount of
extracellular annexin II detected in the media of PC12,
NIH-3T3IR, and NIH-3T3IGF-1R cells but not in
that of untransfected NIH-3T3 cells. Activation of a different growth
factor receptor, the platelet-derived growth factor receptor, did not
produce such results. Tyrphostin AG1024, a tyrosine kinase inhibitor of
insulin and IGF-1 receptor, was shown to inhibit annexin II secretion
along with reduced receptor phosphorylation. Inhibitors of a few
downstream signaling enzymes including phosphatidylinositol 3-kinase,
pp60c-Src, and protein kinase C had no effect on insulin-induced
annexin II secretion, suggesting a possible direct link between
receptor activation and annexin II secretion. Immunocytochemistry
revealed that insulin also induced transport of the membrane-bound form
of annexin II to the outside layer of the cell membrane and appeared to
promote cell aggregation. These results suggest that the insulin
receptor and its signaling pathways may participate in molecular
mechanisms mediating annexin II secretion.
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INTRODUCTION |
Annexin II
(AII)1 belongs to a family of
calcium-dependent phospholipid-binding proteins that are
expressed in diverse tissues and cell types (1). It is found in cells
as a 36-kDa monomer and a 94-kDa heterotetramer (AII94)
made of two 36-kDa monomers (AII36) and two molecules of an
11-kDa (AII11) S100 protein (2). Like all annexins, AII
contains a conserved protein core domain that comprises four repeated
segments of about 70 amino acids each and is resistant to limited
proteolysis. The N terminus of AII contains, as identified by both
in vivo and in vitro studies, a serine
phosphorylation site (Ser-25) for protein kinase C, a tyrosine
phosphorylation site (Tyr-23) for pp60-Src, and a binding site
for AII11 (2-5). Phosphorylation of AII by protein kinase C at this domain has been shown to regulate interaction of the AII
heavy chain with the AII light chain (6, 7) and to influence aggregation of chromaffin granules and lipid vesicles (8, 9), which may
play a role in membrane trafficking events such as
Ca2+-dependent exocytosis (10, 11). On the
other hand, tyrosine phosphorylation of AII in vitro by
pp60c-Src has been shown to play a negative regulatory role in AII's
binding to and bundling of F-actin and formation of the annexin II
heterotetramer complex with plasma membrane (12). The C-terminal domain
of AII contains binding sites for Ca2+, phospholipids, and
F-actin (8).
Initially identified as an intracellular molecule, AII has been
implicated in the regulation of a variety of cellular processes including Ca2+-evoked exocytosis. AII is highly enriched in
chromaffin granule preparations, is able to aggregate chromaffin
vesicles in a Ca2+-dependent manner, and
partially restores the secretory response in permeabilized chromaffin
cells (14). AII-regulated exocytosis appears to be associated with
formation of the heterotetramer AII94. AII has also been
suggested to play a role in endocytosis. It is associated with
endosomal membranes and is one of the few proteins transferred from a
donor to an acceptor endosomal membrane in an in vitro
fusion assay (15). The binding of AII to endosomes appears to be
Ca2+-independent but requires an intact N-terminal domain
(16). Other intracellular functions that involve AII include
immunoglobulin transport (17) and ion channel activity (18).
In addition to its intracellular functions, AII is secreted into the
extracellular environment in both soluble and membrane-bound forms
(19). Although the detailed functions of extracellular AII are not
fully understood, AII is known to interact with matrix proteins and
specific proteases to regulate plasminogen activation, cell migration,
and cell adhesion (20, 21). For example, AII acts as a receptor for the
secreted serine proteases plasminogen and tissue plasminogen activator
on the endothelial cell surface and thereby triggers generation of
plasmin (20, 22). In addition, through interaction with extracellular
matrix proteins such as tenascin-C (23) or certain collagens (24), AII
appears to play a role in mediating cell focal adhesion, migration, and
mineralization of growth plate cartilage. Furthermore, extracellular
and membrane-bound AII may play a significant role in tumor invasion
and metastasis. On the surface of metastatic lymphoma cells, AII
enhances adhesion of these cells to liver sinusoidal endothelial cells
(21). On the surface of tumor cells, the AII heterotetramer interacts
with cathepsin B, a cysteine protease that is secreted into the
extracellular environment and plays a prominent role in tumor
development and invasion (25, 26). Interaction of AII with cathepsin B
may facilitate a proteolytic cascade in the extracellular matrix that selectively degrades extracellular matrix proteins (27).
Because AII lacks a signal peptide and cannot be secreted through
conventional endoplasmic reticulum secretory pathways, the mechanism(s)
by which AII is secreted are currently unknown. Identification of
molecules that participate in transporting AII to the extracellular matrix will be useful for understanding the role of AII in adhesion and
mineralization. It was recently reported that Tyr phosphorylation sites
located on the N terminus of AII can be phosphorylated by the insulin
(IR) and the IGF-1 receptor (28, 29). In this study we report on the
involvement of the IR signaling pathway in regulation of AII secretion.
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EXPERIMENTAL PROCEDURES |
Maintenance of Cell Culture--
Normal NIH-3T3 cells
(nontransfected mouse fibroblasts) and NIH-3T3 cells stably transfected
with human insulin receptor (NIH-3T3IR) or insulin-like
growth factor-1 receptor (NIH-3T3IGF-1R) (30, 31) were
routinely cultured in Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (ICN Biomedical,
Inc.) in 95% humidified air and 5% CO2 at 37 °C. Rat PC12 cells (American Type Culture Collection, Manassas, VA) were maintained in poly-L-lysine (Sigma)-coated (10 µg/ml) T25
plastic flasks in Dulbecco's modified Eagle's medium supplemented
with 10% heat-inactivated horse serum (Invitrogen), 5% fetal bovine serum (Hyclone Laboratories) and penicillin (100 units/ml) and streptomycin (100 µg/ml). For immunocytochemistry experiments, cells
were cultured on poly-L-lysine-coated 10-mm glass
coverslips (Carolina Biomedicals, Inc.).
Treatment of Cells with Insulin, Platelet-derived Growth Factor
(PDGF), and Different Inhibitors--
Once NIH-3T3,
NIH-3T3IR, NIH-3T3IGF-1R, and PC12 cells were
over 95% confluent in culture dishes, they were "starved" in
serum-free Dulbecco's modified Eagle's medium overnight. The culture
medium was replaced with fresh medium at about 15 min prior to insulin treatment. Cells were treated with different concentrations of insulin
or PDGF (100 ng/ml) at 37 °C for 3 min for most experiments except
for the time course experiment in which cells were treated with 100 nM insulin for various times ranging from 0 to 60 min. When
treated with different inhibitors, cells were preincubated with 50 µM IR tyrosine kinase inhibitor tyrphostin AG1024 (Alexis Biochemicals) for 30 min, 100 nM PI 3-kinase inhibitor
wortmannin (Sigma) for 90 min, PP1 (kindly provided by Dr. Anthony
Bishop of Princeton University), and bisindolylmaleimide I (BiSM-1;
Alexis Biochemicals) for 15 min. After preincubation, cells were given another dose of each inhibitor immediately prior to insulin treatment. At the end of the insulin or PDGF stimulation, the culture media were
rapidly transferred to labeled tubes, to which a protease inhibitor
mixture (Sigma) was added to a final concentration of 1%. Cells were
briefly rinsed with precooled PBS and frozen by placing the dishes in
liquid nitrogen. Cells were then thawed on ice and harvested in 200 µl of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate)
containing 1 mM vanadate and 1% protease inhibitor mixture
(Sigma). After incubation on ice for 40 min, the cell lysate was
centrifuged at 8,000 rpm for 3 min at 4 °C to pellet the remaining
cellular debris. The supernatants were collected and kept at
70 °C
for further biochemical experiments.
Immunoprecipitation and Immunoblotting--
The interaction of
AII with IR and IGF-1R was assessed by coimmunoprecipitation. In these
experiments, IR or IGF-1R was immunoprecipitated from the cell lysate
by incubating an equal amount of lysate (200 µg) with either anti-IR
or anti-IGF-1R (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
antibody in lysis buffer at 4 °C on a rotating wheel overnight,
followed by the addition of protein A-agarose and incubation for
another 2 h. The immunocomplex was washed two times with lysis
buffer and one time with washing buffer (20 mM Tris, pH
7.4, 150 mM NaCl). After boiling in 30 µl of SDS-PAGE sample buffer for 10 min, samples were resolved on a 4-20% SDS-PAGE gel (Novex, Inc.) and transferred onto a nitrocellulose membrane (Schleicher & Schuell). Following incubation with PBST (PBS plus 0.1%
Tween 20) containing 5% low fat milk to block nonspecific binding
sites, the membrane was incubated with an anti-AII antibody (Santa Cruz
Biotechnology) at 4 °C overnight and then with a secondary antibody
(Sigma). The immunoreactive signal was revealed in a chemiluminescent
process using an ECL reagent (Pierce). To verify the protein-protein
interaction, AII was also immunoprecipitated with anti-AII antibody,
and the coprecipitated proteins were identified on a Western blot with
anti-IR, anti-IGF-1R, or anti-phosphotyrosine antibody, respectively.
In addition, the amount of protein immunoprecipitated by their own
primary antibodies was routinely measured on Western blots.
Measurement of Protein Phosphorylations of IR, IGF-1R, Atk,
Erk1/2, and AII36--
To detect the activation of IR and
IGF-1R in the cells treated with insulin, the cell lysate was
immunoprecipitated with an anti-phosphotyrosine antibody (Py20; Santa
Cruz Biotechnology Inc.), and the phosphorylated form of IR or IGF-1R
from the immunoprecipitation was revealed by immunoblotting with either
anti-IR or IGF-1R antibody (32). Alternatively, phosphorylation of
these receptors was measured directly on Western blots with an
anti-phospho-IR/IGF-1R antibody (Cell Signaling Technology) that
specifically recognizes the Tyr(P)-1146 of IR and the
Tyr(P)-1131 of IGF-1R. Similar methods were used for detecting
phosphorylations of Akt and Erk1/2 with specific antibodies that
recognize phospho-Akt (Ser-473) or phospho-Erk1/2. Levels of
immunoreactive signals for phosphorylated proteins were then normalized
against that for the total amount of each corresponding protein, which
was revealed with an anti-regular form of the protein. Tyrosine
phosphorylation of AII36 was examined by
immunoprecipitation of AII with an anti-AII36 antibody,
followed by detection of phospho-AII36 on Western blots
with an anti-phosphotyrosine antibody (Py20).
Measurement of Extracellular AII36--
The
overnight culture medium was replaced with fresh medium about 15 min
prior to insulin treatment. Cells were stimulated with 100 nM of insulin at 37 °C for 3 min, and the culture medium was rapidly collected at the end of the reaction. Control cells were
added in the same volume of lymphocyte buffer that was used to dilute
insulin. The collected medium was centrifuged at 5000 rpm for 3 min to
pellet any possible suspended cells or debris in the medium. The
supernatant medium was collected, and the same volume of the medium
from each condition was concentrated using the YM-10 Centricon
(Millipore Corp.). After treatment with SDS-sample buffer, the same
amount of concentrated sample was resolved on a 4-20% gradient
SDS-PAGE gel, and the secreted AII36 was detected on
Western blots by anti-AII36 antibody.
Immunocytochemistry--
The nontreated and insulin-treated
cells were fixed with 4% formaldehyde in PBS (pH 7.4) at room
temperature for 5 min. After the cells were washed three times with
PBS, they were permeabilized with 1% Triton-X100 in PBS (pH 7.4), and
the nonspecific binding sites were blocked with 10% normal horse serum
in PBS (pH 7.4). Cells were then double stained with either
anti-AII/anti-IR or anti-AII/anti-IGF-1R antibodies at room temperature
for 2 h. The dilution of these antibodies was 1:100-200. After
the cells were washed three times with PBS, a secondary anti-goat IgG
conjugated with Texas Red (Vector Laboratories, Inc.) and anti-rabbit
IgG conjugated with fluorescein (Vector Laboratories) were added to the
cells and incubated at room temperature for 1 h in the dark. The
cells were then washed three times, sealed with VECTASHIELD (Vector
Laboratories) and observed under a confocal microscope.
Data Analysis--
The immunoreactive signals from Western blots
were quantified using the NIH Image analyzing program. For experiments
measuring protein phosphorylation using anti-phospho-protein
antibodies, ratios of immunoreactive signals between the phosphorylated
and total amounts of each particular protein were calculated. Signals from insulin-treated cells were converted to the percentage of the
control cells, and values from at least three independent experiments
were subjected to either a two-tailed t test or one-way analysis of variance. p values of less than 0.05 were
considered statistically significant.
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RESULTS |
Coimmunoprecipitation of Annexin II with IR and IGF-1R--
The
NIH-3T3IR and NIH-3T3IGF-1R cells expressed
similar levels of AII36 to those in normal NIH-3T3 cells
(Fig. 1A),
indicating that overexpression of IR or IGF1-R had no effect on the
expression of AII36. In the coimmunoprecipitation
experiment, a substantial amount of AII3
coimmunoprecipitated with both IR and IGF-1R from NIH-3T3IR
and NIH-3T3IGF-1R cells under nonstimulated conditions
(INS
) (Fig. 1B-1). Once cells were treated
with 100 nM insulin (INS +) for 3 min; however, the amount of AII36 "pulled down" by IR or IGF-1R was
markedly reduced (Fig. 1B-1). Statistical analysis of data
from three independent replications indicated a highly significant
effect of insulin treatment (p < 0.001). The reduced
amount of AII36 coimmunoprecipitated with IR and IGF-1R was
not due to a decrease in the amount of IR and IGF-1R precipitated by
the antibodies, since similar amounts of IR and IGF-1R were detected
from the precipitated samples (Fig. 1B-2). This result was
confirmed by a reversed immunoprecipitation experiment, in which an
anti-AII antibody was used during precipitation, followed by detection
of IR and IGF-1R on Western blots with anti-IR or anti-IGF-1R
antibodies. Again, although a similar amount of AII36 was
precipitated by the anti-AII antibody (Fig. 1B-4),
significantly less IR and IGF-1R were coprecipitated with
AII36 after insulin stimulation (Fig. 1B-3). In
addition to the mature IGF-1R, the IGF-1R precursor with a relative
molecular mass of 180-kDa was also "pulled down" with
AII36, suggesting that AII36 interacts with
both the mature receptor and the precursor.

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Fig. 1.
A, expression of AII36 in NIH-3T3 cells.
Similar amounts of cell lysates from NIH-3T3, NIH-3T3IR,
and NIH-3T3IGF-1R cells were resolved on 4-25% SDS
gradient gels followed by detection of AII36 signals with
an anti-AII antibody on a Western blot. No difference in expression of
AII36 was observed across these cell types. B,
interactions of AII36 with IR and IGF-1R. IR and IGF-1R from
nonstimulated and INS-treated NIH-3T3IR and NIH-3T3IGF-1R cells were immunoprecipitated
(ip) with anti-IR and anti-IGF-1R antibody, respectively.
The coprecipitated AII36 was detected with anti-AII
antibody (B-1). The amount of precipitated IR and IGF-1R was
detected on Western blots (ib) with anti-IR or anti-IGF-1R
antibody (B-2). Immunoprecipitation was also performed with
anti-AII antibody followed by detection of the amount of coprecipitated
IR and IGF-1R proteins (B-3) and the amount of
precipitated AII36 (B-4) on Western blots. The
bar graphs in all panels summarize
results from at least three independent replicates (mean ± S.E.;
**, p < 0.001, t test). C,
negative control experiments. 1, cell lysates were
precipitated, respectively, with anti-IR (or anti-IGF-1R), normal
rabbit IgG, and anti-AII36. The precipitated samples were then measured
for AII36 on Western blots. 2, cell lysate was
precipitated with anti-AII, normal goat IgG, and anti-IR (or
anti-IGF-1R) antibodies, followed by detection of IR/IGF-1R on Western
blots. 3, expression of annexin VI in NIH-3T3IR
and NIH-3T3IGF-1R cells. Similar amounts of cell lysates
with and without INS stimulation were resolved in SDS-PAGE.
Immunoreactive signal for annexin VI was measured on Western blots
using a specific anti-annexin VI antibody. 4, the same cell
lysates were subjected to immunoprecipitations with anti-IR or IGF-1R
antibody. The precipitated samples were resolved on SDS-PAGE and
blotted with anti-annexin VI antibody. No annexin VI was detected from
the immunoprecipitated samples. IgG HC, IgG heavy
chain.
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To test the specificity of IR/AII and IGF-1R/AII coprecipitation, we
performed several control experiments in which the normal rabbit and
goat IgG were employed in the immunoprecipitation. As shown in Fig.
1C, in neither NIH-3T3IR nor
NIH-3T3IGF-1R cells was AII36 "pulled down"
by the normal rabbit IgG (Fig. 1C-1). Similarly, neither IR
nor IGF-1R was found in precipitated samples using the normal goat IgG
(Fig. 1C-2). We also examined another member of the annexin
family, annexin VI, on Western blots following immunoprecipitation with
anti-IR and anti-IGF-1R antibodies. As shown in Fig. 1C-3,
annexin VI was expressed in both NIH-3T3IR and
NIH-3T3IGF-1R cells with a higher abundance in the latter.
The expression levels of annexin VI were not affected by INS treatment.
Unlike AII36, no annexin VI was coimmunoprecipitated with
IR or IGF-1R (Fig. 1C-4). These results suggest that AII36
specifically interacts with IR and IGF-1R.
Coimmunoprecipitation of Annexin II with IR and IGF-1R from PC12
Cells--
To test whether coprecipitation of AII36 with
IR and IGF-1R from NIH-3T3IR and NIH-3T3IGF-1R
cells was due to an effect of overexpression of these receptors, we
next examined interactions of AII36 with endogenous IR and IGF-1R in rat PC12 cells. Unlike IR and IGF-1R that were overexpressed in NIH-3T3 cells, IR and IGF-1R were expressed in PC12 cells with markedly different abundance. Fig.
2A shows that levels of
immunoreactive signals for IR from NIH-3T3IR cells and of
IGF-1R from NIH-3T3IGF-1R cells were closely correlated
with amounts of the protein resolved on SDS-PAGE (open
circle, NIH-3T3IR; filled
circle, NIH-3T3IGF-1R). Since only a negligible
level of immunoreactive signals for IR and IGF-1R was detected from
nontransfected NIH-3T3 cells (Fig. 2A, NIH-3T3-1
and NIH-3T3-2), signals detected by anti-IR and -IGF-1R
antibodies from NIH-3T3IR and NIH-3T3IGF-1R
cells should mainly reflect levels of each overexpressed receptor. In
PC12 cells, the relative amount of IGF-1R was about 4 times higher than
that of IR (open square, PC12-IR;
filled square, PC12-IGF-1R). Consistent with that
observed in NIH-3T3IR and NIH-3T3IGF-1R cells,
a substantial amount of AII36 was coprecipitated with IR or
IGF-1R from PC12 cells, which was also significantly decreased (p < 0.001) upon insulin treatment (Fig.
2B). It should be noted that although PC12 cells expressed
high levels of IGF-1R, the relative amount of AII36
coprecipitated with IGF-1R under basal conditions was significantly
lower (p < 0.001) than that with IR.

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Fig. 2.
A, relative abundance of IR and IGF-1R
in PC12 cells. The amount of IR and IGF-1R in PC12 cells were assessed,
respectively, by measuring the immunoreactive signal levels on Western
blots against a concentration curve of the corresponding receptor
overexpressed in NIH-3T3 cells. Open circles, IR
immunoreactive signals in NIH-3T3IR cells;
filled circles, IGF-1R immunoreactive signals in
NIH-3T3IGF-1R cells. Open squares, IR
signals in PC12 cells; filled squares: IGF-1R
signals in PC12 cells; open triangles, IR signals
in nonreceptor-expressed NIH-3T3 cells; filled
triangle, IGF-1R signals in nonreceptor-expressed NIH-3T3
cells. B, coimmunoprecipitation of AII36 with IR
and IGF-1R from PC12 cells. PC12 cell lysates with and without INS
stimulation were subjected to immunoprecipitation with either the
anti-IR or anti-IGF-1R (2 µl/ml) antibody, followed by detection of
AII36 on Western blots. Densitometric values for
coprecipitated AII36 with IR or IGF-1R were normalized,
respectively, against that for each precipitated receptor. The
bar and line graphs (mean ± S.E.) summarize results from at least three repeated experiments. **,
p < 0.001, t test.
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Insulin-induced Increase in Extracellular
AII36--
AII36 is known to be secreted into
the extracellular matrix as both soluble and membrane-bound forms,
although the mechanism(s) and molecular pathway(s) underlying its
secretion have not been identified. In order to understand the
physiological significance of AII/IR and AII/IGF-1R interactions and
subsequent molecular events of the disassociation of AII from these
receptors, we investigated AII secretion from the NIH-3T3,
NIH-3T3IR, NIH-3T3IGF-1R, and PC12 cells. As
shown in Fig. 3A, no
AII36 was detected in the culture medium from untransfected
NIH-3T3 cells under both basal conditions and after insulin
stimulation. In the NIH-3T3IR, NIH-3T3IGF-1R,
and PC12 cells, however, AII36 was found in the culture
medium. The amount of AII36 in the culture media from these
cells was markedly increased following insulin stimulation
(p < 0.001, t test) under those conditions.
Fig. 3, B-1 and B-3, shows activation of IR and
IGF-1R by insulin in a dose-dependent manner as indicated by the extent of tyrosine phosphorylation of the receptors. These receptors were not activated by PDGF. When treated with insulin for 3 min, the amount of extracellular AII36 from
NIH-3T3IR cells was also shown to be insulin
dose-dependent and correlated with phosphorylation levels
of IR. The insulin-induced extracellular AII36 from
NIH-3T3IGF-1R cells, however, remained at a similar level
when insulin concentration was increased from 1 to 100 nM
(Fig. 3B-4). No AII36 was detected in the
culture media from the PDGF-treated NIH-3T3IR and
NIH-3T3IGF-1R cells (Fig. 3, B-2 and
B-4). In addition, no annexin VI was detected in the culture
media under similar conditions (data not shown). When cells were
treated with the IR tyrosine kinase inhibitor AG1024, phosphorylation
of IR and IGF-1R were both inhibited (Fig. 3C-1), along with
a substantial reduction of the amount of AII36 in the
culture media (Fig. 3C-2). However, when cells were treated with the PI 3-kinase inhibitor wortmannin, which abolished the insulin-induced activation of Akt (Fig. 3D), neither the
insulin-induced phosphorylation of IR and IGF-1R nor the
insulin-induced increase in extracellular AII36 was
affected (Fig. 3C). These results suggest that
insulin-induced secretion of AII36 from IR- and
IGF1R-overexpressed cells as well as PC12 cells.

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Fig. 3.
A, insulin-induced changes in the amount
of extracellular AII36. The NIH-3T3, NIH-3T3IR,
NIH-3T3IGF-1R, and PC12 cells were treated with (+) or
without ( ) insulin at 37 °C for 3 min. AII36 from the
concentrated culture medium was dissolved on SDS-PAGE and detected on
Western blots. No AII36 secretion was observed in normal
NIH-3T3 cells. Substantial increases in AII secretion were detected in
NIH-3T3IR, NIH-3T3IGF-1R, and PC12 cells after
insulin stimulation. The bar graphs indicate data
(mean ± S.E.) from three replicates (p < 0.001, t test). B, dose-dependent effect of
insulin on Tyr phosphorylation of IR/IGF-1R and secretion of AII36. In
1 and 3, NIH-3T3IR and
NIH-3T3IGF-1R cells were treated with different
concentrations of insulin or 100 ng of PDGF at 37 °C for 3 min. The
Tyr phosphorylation extent of IR and IGF-1R was measured by
immunoprecipitating the phosphorylated receptors with an
anti-phosphotyrosine antibody. The precipitated phospho-IR and -IGF-1R
were then detected on Western blots with anti-IR or IGF-1R antibody. In
2 and 4, the culture medium was collected after
treatment with different insulin concentrations, and the secreted
AII36 was detected on Western blots with an anti-AII36
antibody. C, effect of tyrphostin AG1024 and wortmannin on
Tyr phosphorylation of IR, IGF-1R, and AII36 secretion. The
NIH-3T3IR and NIH-3T3IGF-1R cells were
pretreated with AG1024 or wortmannin prior to insulin stimulation. The
Tyr phosphorylations of IR and IGF-1R (3C-1) as well as the
extracellular AII36 (3C-2) were measured as described for
B. INS, insulin; wtm, wortmannin;
AG1024, tyrphostin AG1024; P-IR, phosphorylated
IR; P-IGF-1R, phosphorylated IGF-1R. D, effect of
wortmannin on the insulin-induced phosphorylation of Akt. The
NIH-3T3IR and NIH-3T3IGF-1R cells were treated
with or without 100 nM insulin in the presence or absence
of 100 nM wortmannin. The insulin-stimulated
phosphorylation of Akt was detected on Western blots with an
anti-phospho-Akt antibody (3D-1). The total amount of Akt
was measured with an anti-regular Akt antibody (3D-2).
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Time Course of Insulin Effects on Receptor Phosphorylation, Protein
Interaction, and AII Secretion--
A time course study of insulin was
performed to further investigate the insulin-induced AII secretion and
the receptor/AII association. NIH-3T3IR and
NIH-3T3IGF-1R cells were treated with 100 nM
insulin for various lengths of time, and phosphorylation of IR and
IGF-1R was examined on Western blots by an anti-phospho-IR/IGF-1R
antibody. As shown in Fig. 4A,
insulin triggered a rapid phosphorylation of IR and IGF-1R at 3 min after treatment. The phosphorylation of IR remained at similarly
high levels for as long as 60 min after treatment, whereas phosphorylation of IGF-1R showed a time-dependent increase
that peaked at 30 min post-treatment and remained high at 60 min
post-treatment. Whereas the amount of AII36 coprecipitated
with IR and IGF-1R was markedly decreased at 3 min after insulin
treatment, it returned to the control level 15 min after insulin
treatment (Fig. 4B). Secretion of AII36 from
NIH-3T3IR and NIH-3T3IGF-1R cells was detected
at all times examined after insulin stimulation, although it was
slightly reduced from 30 min onward after insulin treatment (Fig.
4C).

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Fig. 4.
A, time course effects of the
insulin-induced receptor phosphorylation. The NIH-3T3IR and
NIH-3T3IGF-1R cells were incubated with 100 nM
insulin, and the reactions were terminated at different times ranging
from 0 (negative control) to 60 min post-treatment. The insulin
stimulated a prolonged receptor phosphorylation measured by an
anti-phospho-IR/IGF-1R antibody on Western blots (4A-1). The
extent of phosphorylation of IR and IGF-1R was assessed by calculating
the ratio of the phospho-IR/IGF-1R to the total amount of each receptor
measured by a regular anti-IR or -IGF-1R antibody (4A-2).
B, time course effects of receptor/AII interaction after
insulin stimulation. Cells were treated with insulin as described for
A, and the receptor/AII36 interaction was
examined as described in the legend to Fig. 1B.
C, time course effects of the insulin-induced secretion of
AII. After treatment with insulin as described for A, the
extracellular AII36 from both NIH-3T3IR and
NIH-3T3IGF-1R cells was measured as described in the legend
to Fig. 3A. In all panels the bar
graphs summarize data from four replications (mean ± S.E.; **,
p < 0.001; one-way analysis of variance).
|
|
Effects of c-Src and Protein Kinase C Inhibitors on
Insulin-stimulated AII Secretion and Interaction--
Since AII is a
major substrate of both pp60c-Src and protein kinase C, to investigate
possible downstream involvement of these protein kinases in
insulin-stimulated AII36 secretion, we applied, respectively PP1, a c-Src inhibitor, and BiSM-1, a cell-permeable protein kinase C inhibitor, to NIH-3T3IR and
NIH-3T3IGF-1R cells 15 min prior to insulin treatment. PP1
inhibited insulin-induced phosphorylation of the extracellular
signal-regulated kinase (Erk1/2), indicating the effectiveness of the
inhibitor (Fig.
5A).
BiSM-1 was also effective in
abolishing phosphorylation of Erk1/2 in human fibroblasts (data not
shown), a downstream event of bradykinin receptor activation (33).
These two inhibitors, however, had no effects on insulin-stimulated
phosphorylation of IR (Fig. 5B) and IGF-1R (Fig.
5C); nor did they affect the subsequent AII secretion (Fig.
5, D and E) and dissociation of AII from
IR and IGF-1R (data not shown). Tyr phosphorylation of
AII36 was assessed by immunoprecipitation of
AII36 with an antibody that interacts with the C terminus
of AII36. The in vivo Tyr phosphorylation of
AII36 was then examined on Western blots with an
anti-phospho-Tyr antibody. An apparent basal level of AII36
Tyr phosphorylation was detected in all cells examined before insulin
treatment, but this Tyr phosphorylation was significantly decreased
(p < 0.001) after insulin stimulation (Fig.
5F). PP1 showed effects on neither the basal
AII36 phosphorylation nor the changes in AII36
phosphorylation under insulin treatment (Fig. 5G). These
results suggest that the insulin-induced AII secretion may be a direct
consequence of receptor activation that does not involve downstream
kinases such as protein kinase C and c-Src.


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Fig. 5.
A, effects of PP1 on insulin-stimulated
Erk1/2 phosphorylation. Cells were treated with or without 100 nM insulin for 3 min at 37 °C in the presence or absence
of different concentrations of PP1. The basal and the
insulin-stimulated Erk1/2 phosphorylation were measured by an
anti-phospho-Erk1/2 antibody (P-Erk1/2). The extent of
phosphorylation was assessed by calculation of the ratio of
phospho-Erk1/2 over the total amount of Erk1/2
(R-Erk1/2). B, effects of PP1 on
insulin-stimulated IR/IGF-1R phosphorylation. Cells were treated under
the same conditions as described for A. The
insulin-activated phosphorylations of IR and IGF-1R were measured by
anti-phospho-IR/IGF-1R antibody (P-IR and
P-IGF-1R). The total amount of IR and IGF-1R was determined
by using the regular anti-IR or anti-IGF-1R antibodies (R-IR
and R-IGF-1R). Ratios of phosphorylated receptors over the
total amount of the receptors were calculated to determine the extent
of receptor phosphorylation. C, effects of BiSM-1 on
insulin-stimulated IR/IGF-1R phosphorylation. Cells were treated with
or without insulin in the presence or absence of different
concentrations of BiSM-1 (0.5-5.0 µM). The resulting
receptor phosphorylation was examined and analyzed as described for
B. D, effects of PP1 on insulin-stimulated
AII36 secretion. Cells were treated as described for
B, and the extracellular AII36 was measured as
described in the legend to Fig. 3A. E, effects of
BiSM-1 on insulin-stimulated AII36 secretion. Cells were
treated as described for C, and the extracellular
AII36 was measured as described in the legend to Fig.
3A. F, changes in AII36 tyrosine
phosphorylation after insulin stimulation. AII36 from
nonstimulated and insulin-stimulated NIH-3T3IR,
NIH-3T3IGF-1R, and PC12 cells were precipitated by anti-AII
antibody, and its Tyr phosphorylation was detected in immunoblot
(ib) with an anti-phospho-Tyr antibody. G,
changes in AII Tyr phosphorylation after insulin treatment in the
presence of PP1 were measured as described for F. The values
of Tyr phosphorylation signals from each condition were normalized with
the total amount of AII36 detected on Western blot with
anti-AII antibody. In all panels, bar
graphs summarize results from three or four experimental
replicates (means ± S.E.; **, p < 0.001;
t test and one-way analysis of variance).
|
|
Immunocytochemistry Results--
Fig.
6 shows the immunocytochemistry results,
in which cells were double stained with either IR or IGF-1R
(green) and AII36 (red) antibodies.
Images in Fig. 6A show double staining of IGF-1R and AII36 in NIH-3T3IGF-1R cells, whereas
images in Fig. 6B show double staining of IR and AII36 in PC12 cells. Under nonstimulated conditions,
AII36 was homogeneously distributed in the cytosolic
compartment. Prominent colocalization of AII36 with both IR
and IGF-1R was seen in both cells indicated by the yellow
color (Fig. 6, A-1 and B-1). Upon insulin stimulation, a reduction of intracellular AII36
levels was seen in both NIH-3T3IGF-1R and PC12 cells alone
with decreased colocalization of AII36 with IGF-1R and IR
(Fig. 6, A-2 and B-2). In addition, an
insulin-induced increase in the amount of AII36 was
detected in the extracellular space. The extracellular
AII36 appeared to attach to the outside surface of the
membrane, particularly in the NIH-3T3IGF-1R cells (Fig.
6B-2, pointed arrow). Fig.
6B-3 shows the enlarged area of the square in
Fig. 6B-2. Similar results were also observed in
NIH-3T3IR cells (data not shown). Western blotting showed
consistent findings that insulin stimulation induced a reduction in
intracellular AII36 in a dose-dependent manner
(Fig. 6C).

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Fig. 6.
Immunocytochemistry staining performed in the
NIH-3T3IGF-1R and PC12 cells. A,
NIH-3T3IGF-1R cells were stained with anti-IGF-IR
and anti-AII antibodies before (1) and after (2)
insulin stimulation. The immunoreactive signals were revealed by the
addition of the secondary antibodies labeled with different fluorescent
dyes. The green fluorescence represents IGF-1R signals and the red
fluorescence represents AII36 signals. The
yellow indicates colocalization of IGF-1R and AII36. The
arrows in 2 point to AII bound to the outside
surface of the cell membrane after insulin stimulation. 3,
an enlargement of field indicated by the square in
2. B, PC12 cells were stained with anti-IR and
anti-AII36 antibodies before (1) and after
(2) insulin stimulation. The IR and AII36 signals were
revealed with fluorescent secondary antibodies. Green, IR
signals; red, AII36. C, changes in
intracellular AII36 after insulin stimulation. PC12 cells were treated
with different concentrations of insulin. The cell lysates were
resolved on SDS-PAGE, and the amount of intracellular AII36
was measured on Western blots with an anti AII36 antibody.
The bar graph represents results from three
replicates. **, p < 0.001.
|
|
 |
DISCUSSION |
Although it has long been known that AII36 can be
secreted into the extracellular compartment, the molecular mechanisms
underlying secretion of AII36 are unknown. Our results show
that insulin stimulation markedly increased the amount of extracellular
AII36, suggesting a link between IR/IGF-1R signaling and
AII36 secretion. AII36 is known as one of the
major substrates of c-Src protein-tyrosine kinase (3, 12) and is also
involved in signaling events downstream from IR (27). Under
nonstimulated conditions, AII36 was associated with IR and
IGF-1R as indicated by coimmunoprecipitation of AII36 with
IR and IGF-1R from NIH-3T3IR, NIH-3T3IGF-1R,
and PC12 cells. Stimulation of cells with insulin markedly reduced association of AII36 with these protein-tyrosine kinases
along with decreased tyrosine phosphorylation of AII36. In
the PC-12 cells, although IGF-1R was shown to be 4 times as abundant as the IR, a similar amount of AII was coprecipitated by both IR and
IGF-1R antibody (Fig. 1, D-1). This may be due to the
possibility that only a proportion of the receptors were associated
with AII36 among the precipitated IGF-1R receptors from
PC12 cells. Such factors as differences in binding affinity to AII
between IR and IGF-1R in PC12 cells may also account for the observed results.
The insulin-stimulated dissociation of AII36 from IR or
IGF-1R was shown to be a rapid but temporary event, since the amount of
AII36 "pulled down" with the receptors returned to the
control level 15 min after insulin stimulation, although
phosphorylation of IR and IGF-1R lasted as long as 60 min after insulin
stimulation. It should be noted that, in addition to the mature IGF-1R
-subunit, AII36 was also associated with the IGF-1R
precursor protein (Fig. 1B-3), which is a single polypeptide
that contains
- and
-subunits of the receptor with a relative
molecular mass of 180 kDa. IGF-1R precursor has been known to exhibit
both ligand-binding ability and tyrosine kinase activity (34, 35). The
insulin-stimulated IGF-1R precursor phosphorylation was also observed
in the present study (Fig. 3C-2). Like the mature receptors,
however, the physiological significance of the AII/IGF-1R precursor
interaction remains to be determined.
Interestingly, the dissociations of AII36 from IR and
IGF-1R were correlated with the insulin-stimulated increases in
extracellular AII36 and a reduction in intracellular
AII36. The extracellular AII36 detected in the
culture medium was not due to the contamination of intracellular
AII36, since all culture media had been carefully centrifuged to remove any suspending cells before concentration. Furthermore, extracellular AII36 was not seen in the
nontransfected NIH-3T3 cells, which expressed the same amount of AII as
NIH-3T3IR and NIH-3T3IGF-1R cells.
The increases in the extracellular AII36 were insulin
dose-dependent (particularly in NIH-3T3IR
cells) and were closely correlated with the tyrosine phosphorylation levels of IR and IGF-1R, an indication of the receptors' activation. The fact that a similar amount of AII was detected in the extracellular compartment of NIH-3T3IGF-1R cells after 1 and 100 nM insulin treatment might suggest that secretion of
AII36 saturates at the activation level of IGF-1R in
response to 1 nM of insulin. Alternatively, as shown by the immunocytochemistry results, a part of AII36 secreted from
NIH-3T3IGF-1R cells under 100 nM insulin
stimulation binds to the outer layer of the plasma membrane, which may
have offset the amount of unbound AII36 detected in the
culture medium. Inhibition of IR and IGF-1R kinase activities by AG1024
significantly reduced the insulin-stimulated extracellular
AII36. The fact that a more obvious reduction of extracellular AII36 was seen with IR inhibition compared
with the IGF-1R inhibition suggests that transport of AII to the
extracellular compartment may be more closely associated with activity
of IR. Furthermore, activation of other growth factor receptors such as
the PDGF receptor resulted in no extracellular AII36. All
of these results indicate that production of the extracellular
AII36 is closely correlated with activities of IR and
IGF-1R.
Since extracellular AII36 was detectable at all of the
post-insulin treatment times and since the insulin-stimulated receptor phosphorylation was also persistent, it is not clear whether the detected AII36 after the longer term insulin stimulation
was due to continuous secretion or was from a one-time secretion at an early stage of the stimulation. Given the fact that association of AII
with IR and IGF-1R returned to control levels 15 min after insulin
treatment, it is possible that AII36 is secreted shortly after activation of IR or IGF-1R and stays in the extracellular matrix
until it is degraded by extracellular proteolysis. The reduction of
extracellular AII36 at 30 and 60 min after insulin treatment may reflect such degradation.
In rat-1 fibroblasts overexpressing the human insulin receptor, insulin
has been reported to induce rapid cytoskeletal protein rearrangement
and membrane ruffling that requires activity of PI 3-kinase (36). One
could argue that the increased extracellular AII36 after
insulin stimulation might be a nonspecific result of an insulin-induced
loss of actin stress fibers and membrane ruffling in the IR- or
IGF-1R-overexpressed cells. To test this possibility, we treated cells
with the PI 3-kinase inhibitor wortmannin, which has been shown to
prevent insulin-induced stress fiber breakdown (36). Treatment of cells
with wortmannin did not reduce the insulin-stimulated increase in
extracellular AII36 but completely abolished the
insulin-induced activation of Akt (Fig. 2D), another insulin-dependent molecular event downstream of PI 3-kinase
(13, 37). These results suggest, therefore, that the insulin-induced extracellular AII36 is not due to cell membrane damage but
rather a receptor activity-driven secretion process.
Although AII36 is known to be a major substrate for protein
kinase C and pp60c-Src tyrosine kinase, inhibition of these two kinases
did not affect the insulin-stimulated AII secretion and changes in its
receptor interaction. It is possible, therefore, that in response to
insulin stimulation, IR and IGF-1R directly regulate AII36
tyrosine phosphorylation through changes in their interaction with
AII36. Thus, secretion of AII36 might be an
immediate cellular event coupled to IR and IGF-1R activation.
The increased extracellular AII36 after insulin stimulation
was not only detected in the culture medium, which should contain the
soluble form of AII36, but also was seen on the outside
layer of the plasma membrane, particularly in the
NIH-3T3IGF-1R cells (Fig. 6A). Given that 1) IR
and IGF-1R were shown to be associated with AII36 but not
with other annexins such as annexin VI; 2) no extracellular annexin VI
was detected under both nonstimulated and insulin-stimulated
conditions; and 3) the increase in extracellular AII36
closely coincided with the reduction of the intracellular AII and the
IR/AII36 and IGF-1R/AII36 shortly after insulin
stimulation, it is tempting to speculate that IR and/or IGF-1R binds to
AII36 and thereby anchors AII to the vicinity of the plasma
membrane. Activation of IR or IGF1-R may regulate the secretion process of AII36 that requires dissociation of the bound
AII36 from the receptors and Tyr dephosphorylation of AII.
However, further research will be required to understand this process
and the molecular mechanisms that underlie secretion of AII and to
verify the role of IR and/or IGF-1R in secretion of AII.
Extracellular AII has been associated with cell adhesion and migration
in both normal and malignant tumor cells through its interactions with
extra matrix proteases and structural proteins. The present results may
shed light on the potential role(s) of the insulin and the insulin-like
growth factor signaling pathways in protein secretion, cell adhesion,
and migration of both normal and malignant cells. Furthermore, given
its roles in these extracellular events, AII may provide a potential
therapeutic target for extracellular matrix-associated pathological
processes such as tumor metastasis.
 |
FOOTNOTES |
*
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.
¶
To whom correspondence should be addressed: Blanchette
Rockefeller Neurosciences Inst., 9601 Medical Center Dr., Academic & Research Bldg., 3rd Fl., Rockville, MD 20850. Tel.: 301-294-7179; Fax:
301-294-7007; E-mail: zhaow@brni-jhu.org.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M210545200
 |
ABBREVIATIONS |
The abbreviations used are:
AII, annexin II;
PDGF, platelet-derived growth factor;
PI, phosphatidylinositol;
IR, insulin receptor;
IGF-1, insulin-like growth factor-1;
IGF-1R, IGF-1
receptor;
PBS, phosphate-buffered saline;
BiSM-1, bisindolylmaleimide
I.
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