(Received for publication, August 5, 1996, and in revised form, November 26, 1996)
From The Hormel Institute, University of Minnesota, Austin, Minnesota 55912
Phorbol esters, which activate isoforms of protein kinase C, are general activators of the transcription factor activated protein 1 (AP-1). The pathway involved in this signal transduction is not very clear. Currently, little is known about whether phosphatidylinositol-3 (PI-3) kinase plays any role in phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced signal transduction. We demonstrate here that TPA not only has markedly synergistic effects on insulin-induced PI-3 kinase activity, but it also can induce PI-3 kinase activity and the PI-3 phosphates by itself. We also found that insulin, a PI-3 kinase activator, enhanced TPA-induced AP-1 trans-activation and transformation in JB6 promotion-sensitive cells. Furthermore, wortmannin and LY294002, two PI-3 kinase inhibitors, markedly decreased AP-1 activity induced by insulin, TPA, or TPA and insulin and inhibited JB6 promotion-sensitive cell transformation induced by TPA or TPA and insulin. Most importantly, constitutive overexpression of the dominant negative PI-3 kinase P85 mutants completely blocked insulin- or TPA-induced AP-1 trans-activation and TPA-induced cell transformation. All evidence from present studies suggests that PI-3 kinase acts as a mediator in TPA-induced AP-1 activation and transformation in JB6 cells.
Phosphatidylinositol-31 (PI-3) kinase plays a central role in a broad range of biological effects (1-4). This enzyme is a dimer composed of a catalytic subunit (P110) and a regulatory subunit (P85) (5). The P85 regulatory subunit has no discernible catalytic activity but possesses two Src homology 2 domains and an Src homology 3 domain (6). A region between the two Src homology 2 domains of P85 binds the NH2 terminus of P110, mediating the constitutive association of the two subunits (6). Binding of P85 to P110 partially activates P110 (7, 8). PI-3 kinase phosphorylates the lipid PI on the 3 position of the D-myoinositol ring, yielding PI-3-phosphate (9). Because the enzyme can also use phosphorylated forms of phosphatidylinositol (PI-4-phosphate and PI-4,5-bisphosphate) as substrates, activation of the PI-3 kinase also leads to the generation of PI-3,4-P2 and PI-3,4,5-triphosphate (5, 9, 10). Previous studies suggested that these PI-3 kinase products are potential second messengers (1, 12, 13).
12-O-Tetradecanoylphorbol-13-acetate (TPA) is not only a
potent tumor promoter in mouse skin (14, 15), but it also induces a
wide range of other biological effects in cultured cells (16). Protein
kinase C (PKC) is well known as a TPA receptor and a
phospholipid-dependent kinase involved in basic cellular
functions, including regulation of cell growth, differentiation, and
gene expression (17, 18). PKC isozymes include more than 11 different
enzymes (,
I,
II,
,
,
,
,
,
,
, and µ).
Previous studies indicated that the TPA-induced activation of AP-1,
NF
B, and other transcription factors in the nucleus is mediated by
the Ras-Raf-1/MAP kinase cascade (18-20). The role of PI-3 kinase in
the TPA-induced signal transduction pathway, however, is not clear,
even though some reports indicated that PI-3 kinase and p21ras
modulate each other's signals (2, 4, 21). For example, overexpression
of activated p21ras in PC12 cells increases PI-3 kinase
activity and stimulates the accumulation of 3
-phosphorylated inositol
lipids in the cells (21). GTP-bound p21ras also directly binds
and activates PI-3 kinase in vitro (21). Expression of
activated PI-3 kinase in NIH 3T3 cells apparently potentiates
p21ras-dependent signaling events (2). Furthermore,
PI-3 kinase products may play an important role in extensive cross-talk
among multiple signaling pathways and regulation of cell function (1,
12, 13). Since both phorbol ester- and insulin-induced activation of
the Ras-Raf/MAP kinase pathway and a combination of insulin and phorbol
ester resulted in a synergistic activation of this pathway (22), we
inquired whether TPA can activate PI-3 kinase and whether activation of
PI-3 kinase is required for TPA-induced signal transduction and cell
transformation. In the present study we used several approaches, which
included a PI-3 kinase activator, two pharmacological PI-3 kinase
inhibitors, and a dominant negative PI-3 kinase inhibitor, to study the
role of PI-3 kinase in TPA-induced AP-1 activation and cell
transformation in the well characterized mouse epidermal JB6
P+ (tumor promotion-sensitive) cells.
The AP-1 luciferase reporter plasmid
(73/+63 collagenase-luciferase) and cytomegalovirus-neo marker vector
plasmid were constructed as previously reported (23); the bovine PI-3
kinase P85 subunit mutant plasmid (
P85) and vector plasmid SR
were as described by Hare et al. (24); agarose conjugated
with monoclonal antiphosphotyrosine antibody Py20 was from Santa Cruz
Biotechnology; fetal bovine serum (FBS) was from Life Technologies,
Inc.; LipofectAMINE was from Life Technologies, Inc.; Eagle's minimal
essential medium (MEM) and wortmannin were from Calbiochem; LY294002
was from BioMol; TPA was from Calbiochem; insulin was from
Sigma; luciferase assay substrate was from Promega;
and [
-32P]ATP was from DuPont NEN.
The JB6 P+ mouse epidermal cell line C1 41 and the stable AP-1 luciferase reporter plasmid transfected mouse epidermal JB6 P+ cell line C1 41-19 (25, 26) were cultured in monolayers at 37 °C in 5% CO2 using Eagle's minimal essential medium containing 5% fetal calf serum, 2 mM L-glutamine, and 25 µg of gentamicin/ml.
Generation of Stable Cotransfectants with AP-1 Reporter and Dominant Negative PI-3 Kinase MutantJB6 P+ cells, C1
41, were cultured in a six-well plate until they reached 85-90%
confluence. We used 2 µg of AP-1 luciferase reporter plasmid and 0.3 µg of cytomegalovirus-neo vector with 6 µg of a dominant negative
mutant of PI-3 kinase P85 subunit plasmid P85 or vector SR
control plasmid DNA and 15 µl of LipofectAMINE reagent to transfect
each well in the absence of serum. After 10-12 h, the medium was
replaced by 5% FBS and MEM. Approximately 30-36 h after the beginning
of the transfection, the cells were trypsinized, and the cell
suspension was seeded into 75-ml culture flasks and cultured for 24-28
days with G418 selection (300 µg/ml). Stable transfectants were
screened by assay of the luciferase activity and Western blotting with
rabbit polyclonal IgG against human PI-3 kinase P85
. Stable
transfected cells,
P85 mass1,
P85 mass2, and AP-1 mass1, were
cultured in G418-free MEM for at least two passages before each
experiment.
Confluent monolayers of JB6 C1
41-19, P85 mass1,
P85 mass2, or AP-1 mass1 cells were
trypsinized, and 5 × 103 viable cells suspended in
100 µl 5% FBS and MEM were added into each well of a 96-well plate.
Plates were incubated at 37 °C in a humidified atmosphere of 5%
CO2. Twelve to 24 h later, cells were starved by
culturing them in 0.1% FBS and MEM for 12 h prior to exposure to
TPA or insulin. The cells were exposed to TPA, insulin, and TPA and
insulin for AP-1 induction with or without different concentrations of
wortmannin or LY294002 for 24 h. The cells were extracted with
lysis buffer, and luciferase activity was measured using a luminometer
(Monolight 2010). The results are expressed as the relative AP-1
activity or relative luciferase units (26).
PI-3 kinase activity was assayed
according to the method of Endemann et al. (27). In brief,
JB6 cells, C1 41, P85 mass1, and
P85 mass 2 or AP-1 mass1, were
cultured in monolayers in 100-mm plates. Then cells were incubated in
0.1% FBS and MEM for 24 h and in serum-free MEM for 3-4 h at
37 °C, respectively. TPA (10 ng/ml) with or without wortmannin was
added, and after 20 min at 37 °C, 2.5 µg/ml insulin was added.
After 10 min at 37 °C, the cells were washed once with ice-cold
phosphate-buffered saline and lysed in 400 µl of lysis buffer/plate
(20 mM Tris, pH 8, 137 nM NaCl, 1 mM Mgcl2, 10% glycerol, 1% Nonidet P-40, 1 nM dithiothreitol, 0.4 mM sodium orthovanadate,
1 mM phenylmethylsulfonyl fluoride). The lysates were
centrifuged, and the supernatants were incubated overnight at 4 °C
with 40 µl of agarose beads previously conjugated with the monoclonal
antiphosphotyrosine antibody Py20. The beads were washed twice with
each of the following buffers: 1) phosphate-buffered saline with 1%
Nonidet P-40, 1 mM dithiothreitol; 2) 0.1 M
Tris, pH 7.6, 0.5 M LiCl, 1 mM dithiothreitol;
and 3) 10 mM Tris, pH 7.6, 0.1 M NaCl, 1 mM dithiothreitol. The beads were incubated for 5 min on
ice in 20 µl of buffer 3, and then 20 µl of 0.5 mg/ml phosphatidylinositol (previously sonicated in 50 mM Hepes,
pH 7.6, 1 mM EGTA, 1 mM
NaH2PO4) was added. After 5 min at room
temperature, 10 µl of the reaction buffer was added (50 mM MgCl2, 100 mM Hepes, pH 7.6, 250 µM ATP containing 5 µCi of [
-32P]ATP),
and the beads were incubated for an additional 5 min. The reactions
were stopped by the addition of 15 µl of 4 N HCl and 130 µl of chloroform:methanol (1:1). After vortexing for 30 s, 30 µl from the phospholipid-containing chloroform phase was spotted onto
thin-layer chromatography plates coated with Silica Gel H containing
1.3% potassium oxalate and 2 mM EDTA applied in
H2O:methanol (3:2). Plates were heated at 110 °C for at
least 3 h before use. The plates were placed in tanks containing
chloroform:methanol:NH4OH:H2O (600:470:20:113)
for 40-50 min until the solvent reached the top of the plates. The
plates were dried at room temperature and autoradiographed. The PI-3
phosphate fractions were scraped off and assayed by scintillation counting (27).
JB6 C1 41 cells were cultured in monolayers in 100-mm dishes. The cells were washed three times with phosphate-free MEM and starved in the same medium for 24 h. Before stimulation, the cell medium was changed to fresh phosphate-free MEM for 3-4 h, and then cells were incubated in the same medium with 0.5 mCi/ml [32P]orthophosphate (ICN) for 3 h at 37 °C. TPA (10 ng/ml) was or was not added, and after 20 min at 37 °C, 2.5 µg/ml insulin was or was not added. The cells were washed twice with ice-cold phosphate-buffered saline 10 min later. Then the cells were extracted by addition of 2 ml of methanol, 0.8 ml of chloroform, 0.1 ml of crude brain phosphoinositide (5 mg/ml in CHCl3) and 0.1 ml of [3H]PI-4,5-bisphosphate (10,000 dpm) in CHCl3. Lipids were deacylated by alkaline hydrolysis (28-31). The deacylated products were separated by high-performance liquid chromatography on a Partisil SAX ion exchange column (Alltech) essentially as described by Scheid and Duronio (30). After a 10-min wash with deionized water, the column was eluted with a 20-min linear gradient from 0-0.25 M ammonium phosphate, pH 3.8, followed by a linear gradient to 1 M ammonium phosphate in 50 min, with a flow rate of 1 ml/min. Fractions of 0.5 ml were collected, and radioactivity was determined by scintillation counting after addition of 3 ml of Ecolume (ICN). About 10 µg of ATP was added to each sample to check the reproducibility of retention times, and the eluate was monitored at 254 nm.
Anchorage-independent Transformation AssayInhibition by wortmannin or LY294002 of TPA or TPA and insulin-induced cell transformation was investigated in JB6 P+ cells. Cells (1 × 104/ml) were exposed to TPA or TPA and insulin with or without wortmannin or LY294002 in 1 ml of 0.33% BME agar containing 10% FBS over 3.5 ml of 0.5% BME agar medium containing 10% FBS. The cultures were maintained in a 37 °C, 5% CO2 incubator for 14-21 days, and the cell colonies were scored by the methods described by Colburn et al. (32). The effect of wortmannin or LY294002 on transformation of JB6 cells was presented as a percentage inhibition of cell transformation.
To determine whether PI-3 kinase plays any role
in TPA-induced signal transduction, we first investigated whether TPA
can induce PI-3 kinase activity. We analyzed PI-3 kinase in JB6
P+ cells stimulated by TPA. As shown in Fig.
1, TPA not only had a synergistic effect on
insulin-induced PI-3 kinase activity, but it also induced PI-3 kinase
activity by itself. Induction or enhancement of PI-3 kinase activity by
TPA could be blocked by wortmannin (Fig. 1). To test whether the PI-3
phosphates were increased in the cells stimulated by TPA, we also
measured the products of PI-3 kinase in TPA-treated JB6 cells by HPLC.
The results show that PI-3,4-P2 in JB6 cells was increased
after cells were stimulated by insulin or TPA (Fig. 2).
PI-3-phosphate and PI-3,4,5-triphosphate could not be conclusively
identified.
Inhibition of TPA-induced AP-1 Activity in JB6 P+ Cells by Wortmannin
Since the above results show that TPA could
stimulate PI-3 kinase activity and enhance insulin-induced PI-3 kinase
activity, we hypothesized that PI-3 kinase may be involved in
TPA-induced AP-1 trans-activation. To test this idea, we incubated JB6
P+ cells with wortmannin, a specific inhibitor of PI-3
kinase, for 30 min prior to TPA stimulation. The results showed that a
nontoxic concentration of wortmannin (100 nM) inhibited
AP-1 activity induced by insulin, TPA, or TPA and insulin by 92.0, 50.0, or 57.3%, respectively (Fig. 3).
Inhibition of TPA-induced AP-1 Activity in JB6 P+ Cells by LY294002
Wortmannin inhibits PI-3 kinase by covalently binding
to P110 of the enzyme. Unlike wortmannin, LY294002 inhibits PI-3 kinase by competing with ATP for its substrate binding site (33). We therefore
used this other PI-3 kinase inhibitor, LY294002, to determine the role
of the PI-3 kinase in AP-1 trans-activation induced by insulin or TPA.
The results showed that LY294002 inhibited AP-1 activity induced by
insulin or TPA in a dose-dependent manner (Fig.
4).
Insulin Induces AP-1 Trans-activation and Enhances TPA-induced AP-1 Activity
Previous studies demonstrated that insulin or TPA
induced partial activation of the extracellular signal-regulated
protein kinase, whereas a combination of insulin and TPA resulted in a synergistic activation of the extracellular signal-regulated protein kinase in Rat-1 HIR cells (22). To investigate whether insulin could
induce AP-1 or promote TPA-induced AP-1 activity in JB6 cells, we
exposed JB6 P+ cells to insulin, TPA, or TPA and insulin.
The results showed that insulin could markedly induce AP-1 activity and
increase TPA-induced AP-1 activity in JB6 P+ cells in a
0.1% FBS medium culture system (Fig. 5A).
The induction of AP-1 activity and the increase in TPA-induced AP-1
activity by insulin occur in a dose-dependent manner (Fig.
5B). At a serum concentration higher than 0.25%, the
increase of TPA-induced AP-1 activity by insulin could not be observed
(Fig. 6).
Overexpression of the Dominant Negative PI-3 Kinase P85
Since significant
inhibition of insulin- or TPA-induced AP-1 activity was achieved by
using both PI-3 kinase inhibitors, wortmannin and LY294002, PI-3 kinase
may play a critical role in TPA- or insulin-induced AP-1 activation.
The dominant negative mutant of PI-3 kinase, P85, has been shown to
be a specific inhibitor of PI-3 kinase in Chinese hamster ovary cells
(24). To specifically block PI-3 kinase and to test the role of PI-3
kinase in TPA-induced AP-1 activation in JB6 cells, the dominant
negative mutant of the PI-3 kinase regulatory subunit P85 plasmid and
AP-1 reporter was cotransfected into JB6 cells by using a LipofectAMINE
kit. Three stable mass cultures, two (
P85 mass1 and
P85 mass2)
from cotransfection AP-1 reporter and SR
P85 plasmids and one
(AP-1 mass1) from cotransfection with AP-1 reporter and vector SR
, were established by G418 selection (34). To determine whether overexpression of the dominant negative PI-3 kinase protein blocks TPA-
or TPA- and insulin-induced PI-3 kinase activity, we also tested the
PI-3 kinase activity in
P85 mass1 and mass2 cells induced by
insulin, TPA, or TPA and insulin. The results show that the PI-3 kinase
activity induced by insulin, TPA, or TPA and insulin was completely
blocked by expression of the dominant negative PI-3 kinase protein
(Fig. 7). Furthermore, overexpression of the dominant
negative PI-3 kinase protein in
P85 mass1 and
P85 mass2 cells
blocked insulin- or TPA-stimulated AP-1 activity completely compared
with that in AP-1 mass1 cells in all the sample points of the time and
dose courses studied (Figs. 8 and
9).
Enhancement by Insulin of TPA-induced JB6 P+ Cell Transformation
Since our previous results and other studies
demonstrated that induced AP-1 activity is important and required for
cell transformation, we tested whether insulin could induce
transformation or promote TPA-induced transformation. The results
showed that insulin could not induce JB6 P+ cell
transformation alone; however, it markedly increased the TPA-induced
JB6 P+ cell transformation rate (Fig.
10).
Inhibition by Wortmannin or LY294002 of JB6 P+ Cell Transformation Induced by TPA and TPA plus Insulin
As shown in Fig. 11, wortmannin and LY294002 inhibited not only TPA- plus insulin-induced JB6 P+ cell transformation but also TPA-induced JB6 P+ cell transformation. This inhibition is in a similar dose range as that observed for the inhibition of PI-3 kinase activity and the inhibition of AP-1 trans-activation.
Blocking of TPA-induced JB6 Cell Transformation by Overexpression ofWe further explored whether the transfectants
expressing the dominant negative PI-3 kinase mutant could repress
TPA-induced transformation. Fig. 12 summarizes the
results of these studies. The TPA-induced transformation in two stable
mass transfectants, P85 mass1 and
P85 mass2, were almost totally
blocked, whereas the AP-1 mass1 cells showed a high frequency of the
transformation rate with exposure to TPA.
Our present studies demonstrate that PI-3 kinase is a crucial
mediator of TPA-induced cell transformation and AP-1 trans-activation in JB6 cells. TPA alone could induce PI-3 kinase activity and increase
the level of PI-3,4-P2 in JB6 cells. More interestingly, TPA and insulin synergistically induced PI-3 kinase activity. Insulin,
a strong PI-3 kinase activator, enhanced TPA-induced AP-1 activation
and cell transformation. Furthermore, wortmannin and LY294002, two
different kinds of PI-3 kinase inhibitors, which inhibit PI-3 kinase by
interfering with P110 and P85, respectively, inhibit TPA-induced AP-1
activation as well as cell transformation. More convincingly, TPA- and
TPA- and insulin-induced AP-1 activation and TPA-induced cell
transformation could be blocked completely by overexpression of the
dominant negative PI-3 kinase P85 mutants in all time courses and dose
responses studied. In contrast, we found previously that wortmannin or
LY294002, as well as overexpression of P85, had no significant
inhibitory effect on JB6 cell proliferation and UV-induced AP-1
activity (34). These observations suggest that the inhibition of AP-1
activation and cell transformation occurred through blocking PI-3
kinase activity but not because of any cytotoxic effect.
Activation of PKC requires both association with the membrane and a
number of activators and cofactors, the requirements for which differ
for each isozyme (35). Thus, PKCs are grouped into three major classes:
conventional PKC isoforms, such as ,
I,
II, and
; novel
PKCs, including
,
,
, and
; and atypical PKCs, represented
by the
and
isozymes of PKC (35, 36). Activation of atypical
PKCs could be carried out by either the PI-3 kinase pathway or the
ceramide pathway (37, 38). Conventional PKCs are activated by
diacylglycerol in a Ca+-dependent manner. In
contrast, activation of novel PKCs is Ca+-independent (35,
36). In addition to the natural activator, conventional PKCs and novel
PKCs are also activated with high specificity by TPA (17). For this
reason, TPA is often used in the study of the mechanisms of
conventional PKC and novel PKC activation and their function. Most of
the previous studies have addressed the regulation of phosphorylation
of the insulin receptor by PKC in PKC isozyme-transfected cells (39,
40). Overexpression of PKC isozymes
,
I,
, and
did not
affect expression of the insulin receptor or insulin-stimulated
tyrosine phosphorylation of the receptor. However, in response to
phorbol esters, cells expressing PKC
,
, and
I but not
exhibited 3-4-fold higher levels of insulin receptor (IR)
phosphorylation. This TPA-stimulated IR phosphorylation inhibits the
activation of the insulin receptor kinase and the insulin-induced PI-3
kinase activity as well as the tyrosine phosphorylation of Shc (39,
40), but this inhibition is not observed in the cells containing only
the endogenous levels of PKC (40). In the present study, we
demonstrated that TPA induces a low level of PI-3 kinase activity and
has significant synergistic effects with insulin on activation of PI-3
kinase in mouse epidermal JB6 cells. The reason for the difference
among data from different cells may be due to different levels of
endogenous PKC in the various cell types studied as well as differences
in ratios of various PKC isozymes present in different cells. The ratio
of PKC:IR in different cells may be another reason for these differences.
Several studies suggested that the PI-3 kinase and its products
PI-3,4-P2 and PI-3,4,5-triphosphate are important
regulators of cell proliferation and c-fos gene expression
(1-3). The introduction of the NH2-terminal Src homology 2 domain of the P85 subunit of PI-3 kinase into cells abrogates the
insulin- or IGF-1-stimulated DNA synthesis and prevents insulin
stimulation of c-Fos protein expression (2, 3). The microinjection of a
dominant-negative p21ras mutant or anti-Ras antibody inhibited
insulin-induced DNA synthesis (3). A constitutively activated mutant
P110 induced transcription from the Fos promoter; coexpression of
dominant negative Ras blocked this response (2). Other studies have
shown that PI-3,4-P2 and PI-3,4,5-triphosphate are elevated
in cells transformed by v-abl, v-src, and polyoma
middle T, and decreased levels of these lipids correlate with impaired
cell transformation by mutated forms of these oncogenes (1, 41, 42). It
has been reported that the presence of insulin-like growth factor I
receptor (IGF-IR) is an obligatory requirement for the establishment
and maintenance of the tumor phenotype (43-46). Cells derived from
mouse embryos with a targeted disruption of the IGF-IR gene
(R cells) cannot be transformed by SV40 T antigen or by
an activated and overexpressed Ha-ras, even by a combination
of both, all of which transform very efficiently the corresponding wild
type cells or other 3T3-like cells (47). If a plasmid expressing a wild type human IGF-I receptor cDNA is stably transfected into
R
cells, the cells can be transformed by SV40 T antigen.
This indicates that the defect in transformability is specifically due
to the lack of IGF-IR (46). Substantial evidence has been reported that
PI-3 kinase is a critical component of signaling pathways used by the
cell surface receptors for a variety of mammalian growth factors or
other stimulators (1, 10, 12, 48), especially IR and IGF-IR. Recently,
it was reported that insulin could activate the Ras-Raf/MAP kinase
pathway by interacting and activating its receptors (7). Dhand et
al. (7) suggested that activation of this Ras/MAP kinase pathway
is critical for the effect of insulin on mitogenesis and
c-fos expression. Others found that neither insulin nor
phorbol ester regulation of phosphoenolpyruvate carboxykinase gene
expression requires activation of the Ras/MAP kinase pathway, but PI-3
kinase is required in this event (49). In contrast, Sakaue et
al. (50) demonstrated that neither the Ras/MAP kinase cascade nor
PI-3 kinase may be required for insulin-stimulated glycogen synthase
activation in Chinese hamster ovary cell lines. Evidence from different
groups using different models has confirmed the crucial importance of
AP-1 activity in transformation and carcinogenesis (23, 26, 51, 53).
Our previous results have shown that AP-1 activation is required for
tumor promotion in the JB6 cell model (23, 26, 52, 53). High basal
levels of AP-1 activity appear to be important for the maintenance of tumor phenotypes in the transformed cell line RT101 (52, 53). Since our
primary results (Fig. 1) showed that TPA can induce PI-3 kinase
activity and increase the level of PI-3,4-P2 (Fig. 2), as
well as have a markedly synergistic effect with insulin on PI-3 kinase
activation, a critical question is whether PI-3 kinase plays any role
in TPA-induced AP-1 activation and cell transformation. To test this
hypothesis, we used several approaches. First, we treated cells with
insulin, a very effective PI-3 kinase activator. The results showed
that activation of PI-3 kinase by insulin resulted in a marked increase
in TPA-induced AP-1 activity. Furthermore, we used two kinds of
pharmacological PI-3 kinase inhibitors to block PI-3 kinase. The first
inhibitor used in our study was a fungal metabolite, wortmannin, which
covalently binds to the catalytic subunit P110 of PI-3 kinase and
irreversibly inhibits the enzymatic activity at nanomolar
concentrations (54). The second PI-3 kinase inhibitor used in this
study was LY294002. Unlike wortmannin, LY294002 reversibly inhibits
PI-3 kinase by competing with ATP for its substrate binding site (33).
These two inhibitors markedly inhibited AP-1 activation and cell
transformation induced by TPA or TPA and insulin in a
dose-dependent manner. Finally, we used a dominant negative
mutant of PI-3 kinase,
P85, in our study. This dominant negative
mutant has been shown to specifically block PI-3 kinase activity and
its mediated cell function in intact cells (24, 50). The stable
introduction of a dominant negative mutant of the PI-3 kinase P85
subunit (
P85) into JB6 cells was shown to block PI-3 kinase activity
by insulin, TPA, or TPA and insulin and also to completely block TPA-
or TPA- and insulin-induced AP-1 activity and cell transformation. All results from our experiments indicate that PI-3 kinase is necessary for
TPA-stimulated AP-1 activation and cell transformation in JB6 cells.
Recent reports suggest that PI-3 kinase and p21ras modulate
each other's signals (2, 21, 24). In JB6 cells, TPA alone induced a
low level of PI-3 kinase activity. This TPA-induced low level of PI-3
kinase activity appears to be required for AP-1 activation and cell
transformation. There are several models that may explain these data.
One interpretation is that the cross-talk between PI-3 kinase and
p21ras is important for the TPA-induced Ras-Raf/MAP kinase
cascade leading to the AP-1 activation and cell transformation. Another
interpretation is that there are some growth factors (such as IGF-I)
that exist in the serum used in cell transformation and have a
synergistic effect with TPA on induction of PI-3 kinase activity. The
last possibility is supported by data from Fig. 5, in that the
enhancement of TPA-induced AP-1 activity by insulin was only observed
at a low concentration of serum.
The signaling pathways induced by insulin have been the subject of intense research (3, 22, 24, 49). Insulin is able to bind to both IR and IGF-IR, but affinities of these two receptors for insulin are different. The affinity of IR for insulin is at least 100 times higher than that of IGF-IR (11). Insulin binding to these two receptors results in receptor-mediated tyrosine phosphorylation of IRS-1 and Shc. These molecules then function as high affinity binding sites for the P85 subunit of PI-3 kinase, and this interaction subsequently results in the activation of PI-3 kinase. In the present studies, we used 2.5 µg/ml insulin as the optimal concentration of insulin for AP-1 activation and costimulation of cell transformation. In this concentration, insulin may bind to both IR or IGF-IR. To whatever receptor insulin binds, after activation of these receptors and subsequent activation of IRS-1, PI-3 kinase is the downstream target of IRS-1. Therefore, the data from our study, in which insulin enhanced the TPA-induced AP-1 activation and cell transformation, still support the concept that PI-3 kinase is required in TPA-induced AP-1 activation and cell transformation in JB6 cells.
In conclusion, we have used several approaches to study the role of the
PI-3 kinase in TPA-induced AP-1 activation and cell transformation in
JB6 cells. TPA could induce PI-3 kinase, and this induction effect was
synergistically enhanced by insulin. The two pharmacological inhibitors
(wortmannin and LY294002) or the biological inhibitor (P85, a
dominant negative mutant of the PI-3 kinase P85 subunit of PI-3 kinase)
markedly blocked TPA-induced AP-1 activation and cell transformation.
Specific blockage of the events required for cell transformation with
few side effects on normal growth might be a promising target for
cancer prevention and treatment. In fact, inhibiting induced PI-3
kinase and AP-1 activity by a pharmacological inhibitor (wortmannin) or
a dominant negative mutant of PI-3 kinase (
P85) does not seem to
have inhibitory effects on cell growth in JB6 cells. Further
investigation of this topic may elucidate the precise mechanisms
underlying the role of PI-3 kinase in phorbol ester-induced signal
transduction and may thus provide a novel target for the prevention of
carcinogenesis.
We thank Dr. Masato Kasuga for the generous
gift of the bovine PI-3 kinase P85 subunit mutant plasmid P85, Dr.
Vincent Duronio for supplying the protocol for HPLC analysis, and
Jeanne Ruble for secretarial assistance.