(Received for publication, July 2, 1996, and in revised form, October 15, 1996)
From the Hormel Institute, University of Minnesota, Austin, Minnesota 55912
In serum-starved NIH 3T3 clone 7 fibroblasts,
choline phosphate (ChoP) (0.5-1 mM) and insulin
synergistically stimulate DNA synthesis. Here we report that ATP also
greatly enhanced the mitogenic effects of ChoP (0.1-1 mM)
both in the absence and presence of insulin; maximal potentiating
effects required 50-100 µM ATP. The co-mitogenic effects
of ATP were mimicked by adenosine
5-O-(3-thiotriphosphate), adenosine
5
-O-(2-thiodiphosphate), ADP, and UTP, but not by AMP or
adenosine, indicating the mediatory role of a purinergic P2 receptor. Externally added ChoP acted on DNA synthesis without its
detectable uptake into fibroblasts, indicating that ChoP can be a
mitogen only if it is released from cells. Extracellular ATP (10-100
µM) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the presence of ATP or insulin, on
the activity state of p42/p44 mitogen-activated protein kinases, while
in combination these agents stimulated the activity of
phosphatidylinositol 3
-kinase (PI 3
-kinase). Expression of a dominant
negative mutant of the p85 subunit of PI 3
-kinase or treatments with
the PI 3
-kinase inhibitor wortmannin only partially (~40-50%)
reduced the combined effects of ChoP, ATP, and insulin on DNA
synthesis; in contrast, the pp70 S6 kinase inhibitor rapamycin almost
completely inhibited these effects. ATP and insulin also potentiated,
while rapamycin strongly inhibited, the mitogenic effects of
sphingosine 1-phosphate (S1P). Furthermore, even maximally effective
concentrations of ChoP and S1P synergistically stimulated DNA
synthesis. The results indicate that in the presence of extracellular
ATP and/or S1P, ChoP induces mitogenesis through an extracellular site
by mechanisms involving the activation of pp70 S6 kinase and, to a
lesser extent, PI 3
-kinase.
Stimulation of a phosphatidylcholine-hydrolyzing phospholipase C activity has been implicated in the actions of several growth factors; however, in practically all cases 1,2-diacylglycerol was thought to be the growth-regulatory molecule (1-5). Recently, it has been reported that in NIH 3T3 fibroblasts externally added choline phosphate (ChoP)1 stimulated DNA synthesis (6). However, a maximal effect required 20 mM ChoP. These authors (6) also reported some limited uptake of ChoP by these fibroblasts, implying that ChoP acted through an intracellular target. Thus, it may appear logical to assume that a high concentration of external ChoP is needed to elicit mitogenic effects because of its poor penetration through the cell membrane.
Recently, using NIH 3T3 clone 7 cells, we found that the addition of 1 mM ChoP to the incubation medium was sufficient to induce maximal effects on DNA synthesis both in the absence and presence of insulin (7). Since the intracellular concentration of ChoP is already about 0.5 mM (6), it was difficult to envision how extracellular ChoP (at 1 mM level) could further increase it. In an attempt to solve this issue, it was necessary to reexamine, as we did in this work, the uptake of ChoP into NIH 3T3 fibroblast cultures, which are available in our laboratory.
The NIH 3T3 cell line, originally selected from NIH Swiss mouse embryo cultures (8), shows considerable heterogeneity. Recently, Wang's laboratory (9) isolated two genetically stable subclones, termed P-3T3 and N-3T3 cells, which apparently make up the major portion of the original NIH 3T3 cell line. The two subclones are different in many respects; most importantly, the growth of P-3T3 cells is stimulated, while that of N-3T3 cells is inhibited, by phorbol 12-myristate 13-acetate (PMA) as well as by v-Abl, v-Src, and Bcr-Abl tyrosine kinases (9, 10). It is conceivable that since the original NIH 3T3 line was established, a considerable degree of heterogeneity among the existing NIH 3T3 lines has developed. Taking these findings into account, it appeared necessary to examine how these subclones respond to the presence of ChoP in the incubation medium. We therefore extended the studies on the mitogenic actions of ChoP to the P-3T3 and N-3T3 subclones as well.
Should ChoP act through an extracellular target, then it clearly becomes important to determine how this compound can reach an effective concentration in the extracellular space. This led us to investigate possible potentiation of both the efflux and mitogenic action of ChoP by an extracellular effector. Among others, extracellular ATP was considered such a potentiating agent because of its known ability to enhance, although usually only modestly, the mitogenic actions of a variety of growth factors (Ref. 11 and references therein).
In fibroblasts, extracellular sphingosine 1-phosphate (S1P) was shown to stimulate cell proliferation (12, 13) and also to mobilize calcium from internal stores via an inositol trisphosphate-independent mechanism (12, 14). In several cellular systems S1P can also activate specific G protein-coupled cell surface receptors (15, 16), which is often associated with transient increases in cytoplasmic calcium concentration (15). Although ChoP has no effects on the distribution of cellular calcium (17), its mitogenic effects may be modified by calcium. Furthermore, ChoP could affect the function of S1P receptor. For these reasons, it was of interest to determine possible interaction between the mitogenic effects of ChoP and S1P.
Here we report that in NIH 3T3 fibroblasts both extracellular ATP and
S1P greatly enhanced the mitogenic effects of ChoP. Cellular uptake of
ChoP was not detectable. However, ATP caused quantitatively significant
release of cellular ChoP into the medium. The mitogenic effects of ChoP
appear to involve pp70 S6 kinase (pp70s6k) and
phosphatidylinositol 3-kinase (PI 3
-kinase). These results suggest
that in the presence of extracellular ATP and/or S1P, ChoP can be an
important positive regulator of cell growth.
ChoP, choline kinase, ATP, ADP, ADPS, ATP
S,
AMP, adenosine, UTP, rapamycin, thapsigargin, ionomycin, and
Dowex-50W-H+ were bought from Sigma;
2-methylthio-ATP was from RBI Research Biochemicals International;
insulin and bombesin were bought from Boehringer Mannheim; p-TYR
(PY20)-agarose conjugate was bought from Santa Cruz Biotechnology,
Inc.;
(1,2-bis(o-aminophenoxy)ethane-N,N,N
N
-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) was purchased from Calbiochem; [methyl-14C]choline (50 mCi/mmol)
and [methyl-3H]thymidine (85 Ci/mmol) were
from Amersham Corp.; and tissue culture reagents were purchased from
Life Technologies, Inc. [14C]ChoP was prepared from
[14C]choline by a choline kinase-mediated phosphorylation
reaction in a reaction mixture (0.1 ml) containing
[14C]choline (25 µCi), choline kinase (1 unit/ml), ATP
(10 mM), MgCl2 (10 mM), KCl (150 mM), and Tris buffer, pH 9.0 (50 mM). After incubation for 3 h at 22 °C, [14C]ChoP was
separated on Dowex-50W-H+ columns as described below.
PhosphoPlus MAP kinase antibody kit and phosphatidylethanol were bought
from Biolabs and Avanti Polar Lipids, respectively.
NIH clone 7 (clone 7/3T3) fibroblasts transfected with Harvey murine sarcoma virus (18) as well as the parent cell line were obtained from Dr. Douglas R. Lowy (NCI, National Institutes of Health, Bethesda, MD). Cells were cultured continuously in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, penicillin/streptomycin (50 units/ml and 50 µg/ml, respectively), and glutamine (2 mM). The genetically stable P-3T3 and N-3T3 subclones of NIH 3T3 cells, generously provided by Dr. Jean Y. J. Wang (University of California, La Jolla, CA), were maintained as described previously (10).
Labeling of Cellular DNA with [3H]ThymidineNIH 3T3 fibroblasts were first grown in 12-well tissue culture dishes to about 60-70% confluence in the presence of 10% serum, followed by incubations in serum-free medium for 24 h. Fibroblasts were then treated (in serum-free medium) first with ATP for 10 min and then (in the continuous presence of ATP) with ChoP and/or insulin for 16 h. Finally, incubations were continued in the presence of [methyl-3H]thymidine (1.0 µCi/well) for 60 min. The cells were washed twice with phosphate-buffered saline and then four times with 5% trichloroacetic acid. The acid-insoluble material was dissolved in 0.3 M sodium hydroxide, and an aliquot was taken to measure DNA-associated 3H activity in a liquid scintillation counter.
Measurement of Cellular Uptake of [14C]ChoPUntransformed clone 7/3T3, P-3T3, and N-3T3 as well as Ha-Ras-transformed NIH 3T3 fibroblasts were grown in 12-well tissue culture dishes first in the presence of 10% serum and then for 24 h in the absence of serum. Confluent cultures of fibroblasts were then incubated (in serum-free medium) in the presence of [14C]ChoP (855,000 dpm/well; 0.1 mM) in the absence or presence of ATP for up to 10 h in an incubator. At the conclusion of incubations, the incubation medium (0.75 ml) was harvested (added to tubes containing 2 ml of chloroform), and then the cells were rapidly (in 20 s) washed with 5 ml of serum-free medium followed by the addition of 2 ml of ice-cold methanol to the wells. The cells were scraped into methanol, followed by the rapid transfer of methanol extracts to tubes containing 2 ml of chloroform.
Fractionation of choline metabolites was performed on Dowex-50W-H+-packed columns (Bio-Rad Econo-columns; 0.75-ml bed volume) with minor modifications of the procedure described by Cook and Wakelam (19). The initial flow-through (5 ml) along with a following 5-ml water wash contained glycerophosphocholine. ChoP and choline were successively eluted by 18 ml of water and 12.5 ml of 1 M HCl, respectively. The metabolites of [14C]choline were further identified by TLC, and phospholipids were determined as previously indicated (20).
Measurement of [14C]Choline Metabolism in and Release of [14C]ChoP from NIH 3T3 FibroblastsThis was determined as described above, except that fibroblasts were incubated with [14C]choline (1.23 × 106 dpm/well; 50 µM) for 3 h.
Determination of MAP Kinase ActivityCells in 6-well tissue
culture dishes were treated first for 10 min, when appropriate, with
ATP, and then for an additional 5, 15, or 30 min with ChoP, insulin, or
PMA in combinations and at concentrations indicated in the legend to
Fig. 6. Samples for immunoblot analysis were prepared as described
previously (21). Phosphospecific MAP kinase antibody (Biolabs), which
recognizes the tyrosine 204 phosphorylation site, was used to detect
the activated (phosphorylated) forms of p42 and p44 MAP kinases. The Western immunoblotting protocol was performed according to the instructions provided by the manufacturer.
Stable Overexpression of Dominant Negative Mutant of p85 Subunit PI 3
The plasmid SR-
p85, which contains the
dominant negative mutant version of the p85 subunit of bovine PI
3
-kinase, was kindly provided by Dr. Masato Kasuga (Kobe University,
Japan); the details for the construction of the plasmid have been
described elsewhere (22). NIH 3T3 clone 7 cells were co-transfected
with pRBK, a plasmid conferring hygromycin B resistance, and
SR
-
p85 (or the empty vector) using Lipofectin (Life Technologies,
Inc.) as described previously (23). Selection was started by the
addition of 220 µg/ml hygromycin B to the culture medium. After
14-16 days in selection medium, single clones were subcloned by
limited dilution and examined for the presence of dominant negative
mutant p85 mRNA and the protein subunit by RT-PCR and Western
blotting, respectively. For RT-PCR, the total cellular RNA was isolated
from transfected NIH 3T3 cells grown in the hygromycin B selection
medium using the STAT 60 RNA isolation kit (Tel-Test, Inc.). RT-PCR was
then performed using the Access RT-PCR System (Promega) following the procedure recommended by the manufacturer. Each reaction mixture contained 1 µg of RNA template and 50 pmol of upstream and downstream primers, which had the sequence 5
-CCC GAA CTT CCC AGG AAA TCC-3
and
5
-GTC AAT CTC ACG ATA CTC AGC-3
, respectively. Two control reactions,
one with RNA from untransfected NIH 3T3 cells and the other with RNA
template supplied from the kit, were included. The primers were
designed to hybridize the flanking sequences of the deletion in
p85.
After 40 cycles of polymerase chain reaction, the amplified cDNA
products were analyzed on a 1.5% agarose gel.
Serum-starved (24 h) fibroblasts, grown
in 100-mm plastic dishes, were treated for 20 min with agents as
indicated in Fig. 8. PI 3-kinase activity was determined in
anti-phosphotyrosine immunoprecipitates by the method described by
Okada et al. (24, 25) with several modifications. Washed
cells were scraped into ice-cold lysis buffer (1 ml) containing 137 mM NaCl, 20 mM Tris/HCl, pH 7.4, 1 mM MgCl2, 1 mM dithiothreitol, 10%
glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 1% Nonidet P-40.
After solubilization on ice for 10 min, insoluble material was removed
by centrifugation at 15,000 × g for 15 min. PI
3
-kinase enzyme was immunoprecipitated by incubating the lysates with
40 µl of PY20-agarose conjugate overnight at 4 °C. After
successive washings (24), the pellets (on ice) were redissolved in a
buffer (20 µl) containing 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM dithiothreitol, and 1 mM Na3VO4, followed by the addition
of 30 µl of PI 3
-kinase reaction mixture containing 0.5 mg/ml
phosphatidylinositol (dispersed by sonication in a solution containing
50 mM Hepes, 1 mM EGTA, and 1 mM
NaH2PO4), 5 mM MgCl2,
and [
-32P]ATP (100 µM; 5 µCi). The
reactions were carried out in a water bath at 30 °C for 5 min and
then stopped by successive addition of 15 µl of 4 M HCl
and 0.13 ml of chloroform/methanol (1:1, v/v). After phase separation,
the product phosphatidylinositol 3-phosphate was separated on silica
gel G plates impregnated with 1% potassium oxalate as described (25).
After autoradiography, the spots were evaluated by densitometry or by
determining 32P activity in the spots in a liquid
scintillation counter.
Potentiating Effects of ATP on the Mitogenic and Co-mitogenic
Effects of ChoP in Clone 7/3T3 FibroblastsThe addition of ATP (10-100 µM) alone to serum-starved clone 7/3T3
fibroblasts failed to stimulate DNA synthesis over a 16-h incubation
period, but it enhanced, although less than 2-fold, the relatively
small stimulatory effect of insulin (Fig. 1). Since 100 µM ATP was certainly maximally effective, in most of the
following experiments this concentration was used to determine its
possible effects on ChoP and insulin-induced mitogenesis.
Next we examined the ability of ATP, alone and in combination with
insulin, to reduce the effective mitogenic concentration of ChoP. In
the absence of other agents, 1 mM ChoP enhanced DNA synthesis about 10-fold, while 0.5 mM or lower
concentrations of ChoP had much smaller effects or no effects (Fig.
2). In the presence of ATP or ATP plus insulin (500 nM), 1 mM ChoP was capable of stimulating DNA
synthesis about 45- or 215-fold, respectively (Fig. 2). It should be
noted that in the same experiment 10% serum stimulated DNA synthesis
about 160-fold (not shown). Importantly, in the presence of ATP and
insulin even 0.1 mM ChoP, a concentration that may be
physiologically quite relevant (see below), stimulated DNA synthesis
26-fold (Fig. 2). A similar effect in the presence of ATP alone or
insulin alone required about 0.25 or 0.5 mM ChoP, respectively (Fig. 2).
To have a better idea of the mechanism of co-mitogenic ATP effects,
next we determined the effects of several ATP analogues and other
nucleotides on insulin and/or ChoP-induced DNA synthesis; each compound
was used at a 100 µM concentration. AMP and adenosine were totally ineffective, and 2-methylthio-ATP was about 45% as effective as ATP, while ATPS, ADP, ADP
S, and UTP were equally effective, or even more effective, than ATP (Table
I).
|
It was known that
only P-type, and not N-type, cells respond to the cell growth
stimulatory effects of PMA and tyrosine kinase activators (9, 10). Data
in Fig. 3 show that while the responses of these cells
to insulin alone were similar, insulin and ChoP had significant
synergistic effects on DNA synthesis only in the P-3T3 subclone.
ATP was able to significantly enhance the mitogenic effects of both
ChoP alone and ChoP plus insulin in both subclones, although the three
agents together were still at least 3-4 times more effective inducers
of DNA synthesis in the P-3T3 than in the N-3T3 cells (Fig.
4). However, these differences appeared to be mainly due to the poor response of N-3T3 cells to ChoP plus insulin but not to
ATP. The finding that the combined effects of ChoP, ATP, and insulin
were only quantitatively different in the two subclones suggested that
the use of clone 7/3T3 cell population for additional studies on the
combined effects of ChoP and ATP will not lead to misinterpretation of
data.
ATP Stimulates the Release but Not the Cellular Uptake of ChoP in Various Fibroblast Cultures
Previous data reported by another laboratory (6) indicated incorporation of a limited amount of ChoP into NIH 3T3 fibroblasts when it was added to the medium at a high (20 mM) concentration. This is an important issue, because if ChoP acts through an intracellular target, the use of this compound even at 20 mM concentration would be justified. However, should ChoP act through an extracellular target, then questions arise of where extracellular ChoP is derived from and what concentration range this compound might reach in the extracellular space. For these uptake studies, the lowest mitogenically active concentration (0.1 mM) of [14C]ChoP was used, because this is likely to be physiologically relevant, and we also tried to maintain the specific activity of ChoP as high as possible. During a 10-h incubation period, we could detect neither the uptake of [14C]ChoP into the clone 7/3T3, P-3T3, and N-3T3 cultures nor its incorporation into cellular phosphatidylcholine (Table II). Similarly, no uptake of labeled ChoP into clone 7 fibroblasts was observed after incubations for 2, 4, or 6 h (data not shown). In contrast, Ha-Ras-transformed fibroblasts incorporated significant amounts of labeled ChoP, which then was used for phosphatidylcholine synthesis (Table II). Interestingly, in the transformed cells both the uptake and incorporation of [14C]ChoP into phosphatidylcholine were inhibited by ATP (Table II). These results with the transformed cells also indicated that the experimental design used here was suitable to detect incorporation of labeled ChoP into fibroblasts.
|
It is important to add that, both in the absence and presence of ATP, added labeled ChoP remained metabolically very stable in the incubation medium; in the three untransformed cell populations we could not observe any statistically significant degradation of labeled ChoP during a 10-h incubation period (data not shown).
The results shown above made it clear that ChoP must induce mitogenesis
through an extracellular site. Therefore, if ChoP plays a role in
mitogenesis, it should be able to exit the cells in a probably highly
regulated manner. To see if this is the case, next we tested the
possible effect of ATP on the release of ChoP from fibroblasts into the
medium. When clone 7/3T3 fibroblasts were incubated in the presence of
[14C]choline for 4 h, only a relatively small
portion (~6%) of total [14C]ChoP formed was recovered
from the incubation medium (Fig. 5). However, ATP at a
concentration as low as 10 µM doubled the release of
labeled ChoP, while in the presence of 50-100 µM ATP the
incubation medium contained about 20-22% of the total
[14C]ChoP formed during the 4-h period. When treatments
with 100 µM ATP were extended up to 12 h, we
observed that about 40% of the total labeled ChoP formed was present
in the incubation medium (data not shown). This probably reflects both
the inability of ChoP to reenter the cells and the metabolic stability
of ChoP in the incubation medium. In several other cell lines,
including C3H/10T1/2 and Rat1 fibroblasts, MCF-7 human breast carcinoma cells, and JB6 epidermal cells, ATP induced similarly extensive release
of intracellular ChoP (data not shown). The most important conclusion
that can be drawn from these experiments is that the effect of ATP on
ChoP release appears to be a widespread phenomenon. In addition, it is
conceivable that at a relatively high ratio of cell to extracellular
space volume, which is the case in most tissues, ATP can increase the
extracellular concentration of ChoP into the mitogenically active range
of 0.1-0.5 mM.
Effects of ChoP, Insulin, and ATP on MAP Kinase Activity
Initially, insulin stimulates several growth-regulatory
mechanisms involving Shc-Grb2-Sos trimers as well as the various
complexes of insulin receptor substrate 1 with Grb2, PI 3-kinase, Syp, and Nck. In many cell lines the Ras
MAP kinase signal transduction pathway appears to be a major downstream converging point for these
mechanisms (26-28). In contrast, in Swiss 3T3 (29) and Balb/c 3T3
cells (30), insulin alone did not stimulate MAP kinase activity.
Similarly, when we assessed MAP kinase activity by measuring its rate
of tyrosine phosphorylation (31, 32), we observed that in NIH 3T3
fibroblasts insulin had only a very small, nearly undetectable,
transient effect on this enzyme activity between 5 and 15 min of
incubation. At these time intervals, ChoP in combination with either
insulin or ATP also had only very small effects (data not shown).
Reproducible, but still very small, effects of ChoP in combination with
insulin or ATP were observed after 30 min of incubation (Fig.
6); at this incubation time, insulin or ChoP alone had
no visible effects (Fig. 6). In contrast, PMA, which is a less
efficient inducer of DNA synthesis than ChoP plus insulin or ChoP plus
ATP (Table III), greatly stimulated MAP kinase activity (Fig. 6). Furthermore, similar to Balb/c 3T3 fibroblasts (28), PMA and
insulin synergistically stimulated MAP kinase activity (Fig. 6). Thus,
in these fibroblasts only the protein kinase C-dependent mitogenic actions of insulin appear to involve the MAP kinase pathway.
|
Similar to many other mitogens, insulin
also stimulates ribosomal S6 kinases (pp70s6k and
pp90rsk). It is believed that activation of MAP kinase and
pp90rsk are on the same pathway, while activation of
pp70s6k by insulin occurs by a MAP kinase-independent (33)
and PI 3-kinase-dependent mechanism (34). Rapamycin (5 nM), an apparently specific inhibitor of
pp70s6k (34, 35), strongly inhibited the combined mitogenic
effects of ChoP plus ATP and/or insulin (Fig. 7).
Rapamycin also effectively inhibited the mitogenic effects of
platelet-derived growth factor and insulin-like growth factor I (data
not shown). In comparison, a concentration of wortmannin (200 nM) that should have maximal inhibitory effect on PI
3
-kinase (36, 37), had clearly smaller inhibitory effects on ChoP plus
ATP and/or insulin-induced DNA synthesis (Fig. 7). None of these
inhibitors had detectable effects on cell viability as determined by
the trypan blue exclusion assay.
Recent studies suggest that wortmannin may not be a specific inhibitor
of PI 3-kinase (38, 39). To further examine the role of PI 3
-kinase
in the mediation of ChoP effects, we expressed the dominant negative
mutant of the p85 subunit of this enzyme. In each of the five clones
isolated, the ratio between the wild type (0.268 kilobase pairs) and
mutant (0.169 kilobase pairs) subunits was about 1:1, based on the
detection of amplified cDNA. Most studies were performed with one
clone, but in an additional experiment the results were confirmed with
the four other clones. As shown in Table IV, in the
mutant p85 expressor cells the effects of ChoP plus insulin and ChoP
plus ATP as well as the combined effects of the three agents on DNA
synthesis were similarly, but only partially (~40-50%), reduced
compared with the vector control cells. We should note here that for
presently unknown reasons a previous exposure of cells to hygromycin B
decreased the mitogenic effect of ChoP alone, although such treatment
did not seem to affect the mitogenic activities of ATP and insulin in
the presence of ChoP. This effect of hygromycin B is unlikely to affect
interpretation of data, because both the vector control and the mutant
expressor cells were exposed to hygromycin B for the same period of
time. As a positive control, in the mutant p85 subunit expressing cells the mitogenic effect of PMA was almost completely blocked (Table IV).
This result was expected, because previously we showed (40) that in NIH
3T3 cells wortmannin strongly inhibited PMA-induced DNA synthesis.
|
If the mitogenic effects of ChoP plus ATP and ChoP plus insulin
involved PI 3-kinase activity, as suggested by data in Table IV, then
these agents would be expected to increase the lipid kinase activity of
this enzyme. As shown in Fig. 8, insulin alone, and
particularly in combination with PMA or ChoP, significantly enhanced
the formation of phosphatidylinositol 3-phosphate by PI 3
-kinase;
these stimulatory effects ranged from ~3.5- to 6-fold, as determined
by densitometry. Formation of phosphatidylinositol 4-phosphate was not
observed. ChoP and ATP alone had no effects (not shown), but in
combination they enhanced PI 3
-kinase activity nearly 2-fold (Fig. 8).
The combined stimulatory effects of ChoP and insulin were not enhanced
by ATP (Fig. 8). Overall, these data indicated that the ATP- and
insulin-dependent stimulation of DNA synthesis by ChoP is
associated with increased PI 3
-kinase activity.
Since
extracellular S1P stimulates proliferation of fibroblasts (12, 13), it
was of interest to examine a possible relationship between the
mitogenic actions of S1P and ChoP. In NIH 3T3 fibroblasts, 0.5 µM S1P caused an approximately 3-fold increase in DNA
synthesis, and a maximal (~13-fold) stimulatory effect required ~5
µM S1P (Fig. 9). Similar to the mitogenic
effect of ChoP, the stimulatory effects of both lower and higher
concentrations of S1P on DNA synthesis were greatly enhanced by both
ATP and insulin (Fig. 9). Furthermore, S1P-induced DNA synthesis was
also more effectively inhibited by rapamycin than by wortmannin (Fig.
10). More surprisingly, ChoP and S1P synergistically
enhanced, even when both presented at maximally effective
concentrations, DNA synthesis (Fig. 11). Extracellular ATP was able to further increase the synergistic effects
of ChoP and S1P (Fig. 11). It should be noted here that other
phosphate-containing compounds, including ethanolamine phosphate (0.1-2 mM) and creatine phosphate (0.1-5 mM)
had no mitogenic effects.
Possible Role of Calcium in ChoP-induced Mitogenesis
Both S1P (12, 14) and ATP (Ref. 41 and references therein) can cause transient increases in cytoplasmic calcium levels. Although ChoP does not mobilize calcium (17), an increase in intracellular calcium by ATP or S1P may enhance the mitogenic effects of ChoP. If this is the case, then other calcium-elevating agents would also be expected to enhance the mitogenic effects of ChoP. In fibroblasts, bombesin has been shown to raise the level of cytoplasmic calcium as a consequence of increased hydrolysis of inositol phospholipids (42-44). In agreement with data in a previous report (45), a maximally effective concentration (100 nM) of bombesin alone had only a relatively small (~2.5-fold) stimulatory effect on DNA synthesis (Table V). However, bombesin clearly potentiated, although less efficiently than ATP, the mitogenic effect of ChoP (Table V). It is of interest to note that in the presence of ATP, bombesin had no such potentiating effect (Table V), suggesting that ATP and bombesin used, at least in part, the same mechanism to enhance the mitogenic effect of ChoP. A short (10-min) pretreatment with the calcium-mobilizing agents thapsigargin (1 µM) and ionomycin (1 µM), followed by treatment of washed cells with ChoP for 16 h, also led to 1.6-1.8-fold increases in the mitogenic effects of ChoP (data not shown). In another experiment, initially performed in a calcium-free medium, 10 µM BAPTA/AM (a cell-permeable calcium chelator) was added to cells 20 min prior to ATP (100 µM); after an additional 10-min incubation in the presence of ATP, cells were washed and then incubated further in a calcium-containing medium for 16 h in the presence of ATP and ChoP. Under these conditions, ATP enhanced the effect of ChoP (1 mM) on DNA synthesis about 2.8-fold, and BAPTA/AM inhibited the potentiating ATP effect, but not the effect of ChoP alone, by ~40% (data not shown).
|
In this work we have shown that extracellular ATP could greatly
enhance the mitogenic and co-mitogenic effects of ChoP, even when the
latter was present at a maximally effective concentration. Since a
number of P2 receptor agonists, but not AMP or adenosine, mimicked the co-mitogenic effects of ATP, it seems clear that the
effects of ATP were mediated by one of the P2 receptors.
Furthermore, the finding that UTP was also able to enhance the effect
of ChoP suggests the specific role of the P2u receptor,
which is uniquely stimulated by UTP, in addition to ATP and ATPS
(46, 47).
Perhaps the most important aspect of the co-mitogenic ATP effect is that it decreased the mitogenically active concentration of ChoP from about 1 mM to 0.1-0.25 mM. Since in normal fibroblasts the concentration of ChoP is around 0.5 mM (6), it is reasonable to assume that a substantial release of ChoP from these cells could elevate the concentration of ChoP in the extracellular space to the 0.1-0.25 mM range. In this context, the ability of ATP to induce extensive release of ChoP from cells becomes a very important issue, because presently this is the only known mechanism by which ChoP can become a mitogen. Preliminary studies in our laboratory show that after a 4-h treatment period ATP induces massive ChoP release (15-35% of the total cellular pool) in each cell type examined so far, including C3H/10T1/2 fibroblasts, Rat1 fibroblasts, MCF-7 human breast carcinoma cells, and epidermal JB6 cells. Thus, it appears that if once the concentration of ATP in the extracellular space reaches a certain value (10-50 µM), then a sufficient amount of ChoP from cells is probably released to enhance cell growth, an action that is also potentiated by ATP. Accordingly, extracellular ATP is clearly an agent that not only increases the release of ChoP, but at the same time also potentiates the action of ChoP through an extracellular target. Interestingly, despite the large number of papers describing the effects of extracellular ATP, the issues of how much ATP is present in the extracellular space and in the circulation and how ATP molecules can get there are much less studied. However, it is known that ATP can be liberated from cells at the site of a wound, during platelet activation, and by the necrosis of cells during tumor growth (reviewed in Ref. 48). Clearly, more information is needed about the regulation of ATP concentration in the extracellular medium before the true physiological significance of the combined mitogenic effects of ChoP and ATP can be assessed.
While the p42/p44 MAP kinases appear to play key roles in the mediation of mitogenic effects of insulin in many cellular systems (26-28), in NIH 3T3 cells, as in other 3T3 lines (29, 30), these enzymes were significantly activated by insulin only in the presence of PMA. Although in the presence of ChoP insulin slightly activated MAP kinase, these effects were much smaller than that obtained with PMA alone. Collectively, these observations indicate that the mitogenic effects of ChoP, both in the presence of insulin and ATP, are predominantly mediated by a MAP kinase-independent pathway and that only the protein kinase C-dependent mitogenic effects of insulin involve a MAP kinase-dependent pathway.
Previous reports suggested that in fibroblasts p42/p44 MAP kinases are required for mitogenesis (49, 50). However, in Swiss 3T3 fibroblasts, these MAP kinases were less important for the mitogenesis induced by bombesin plus insulin (29). Our results further indicate that stimulation of DNA synthesis through combined treatments with ChoP, ATP, and insulin is possible without involving the MAP kinase-dependent mitogenic pathway.
ChoP, when added in combination with insulin or ATP, stimulated PI
3-kinase activity. In addition, the expression of a dominant negative
mutant of the p85 subunit of PI 3
-kinase or the addition of wortmannin
led to partial inhibition of the combined mitogenic effects of ChoP,
ATP, and insulin. These data indicate that while maximal increase in
DNA synthesis by these agents requires PI 3
-kinase, treatments with
ChoP, ATP, and insulin can also induce mitogenesis by a PI
3
-kinase-independent pathway.
In contrast to wortmannin, rapamycin, a specific inhibitor of
pp70s6k (34, 35), strongly inhibited the mitogenic effects
of ChoP both in the absence and presence of ATP and insulin. These
observations implicate pp70s6k in the mediation of
mitogenic effects of ChoP. The pp70s6k enzyme is a
ubiquitous Ser/Thr kinase that is activated by virtually all known
mitogens and that plays a key role in the progression of cells from
G1 to S phase of the cell cycle (51-54). Full activation of pp70s6k requires several independent stimuli resulting
in multiple phosphorylation of the enzyme (Ref. 55 and references
therein). Activated pp70s6k phosphorylates ribosomal S6
protein, resulting in increased translation of mRNAs containing a
polypyrimidine tract (56). PI 3-kinase is an important regulator of
pp70s6k (34), which directs site-specific phosphorylation
of Thr252 in the catalytic domain of pp70s6k
(57). Thus, the PI 3
-kinase-dependent effects of ChoP may also involve pp70s6k.
Similar to ChoP, extracellular S1P also enhanced DNA synthesis in a strongly ATP- and insulin-dependent manner. While S1P has been shown to stimulate DNA synthesis in fibroblasts (12, 13), this is the first study to reveal its synergistic interaction with ATP and insulin. However, even maximally effective concentrations of ChoP and S1P had strong synergistic effects, indicating important differences in their actions. One clear difference is that S1P, in contrast to ChoP, is an activator of MAP kinases (58). Collectively, these findings suggest three possible mechanisms to explain the synergistic actions of ChoP and S1P. (i) ChoP and S1P may act through two separate sites. In this case, their individual effects may be amplified by a common target along the signal transduction pathway, or the effect of ChoP may be amplified by calcium generated by S1P (as well as by ATP, bombesin, and possibly other calcium-elevating agents). (ii) ChoP and S1P may act through separate sites on pp70s6k (both ChoP and S1P) and MAP kinase (only S1P) activities, resulting in synergistic activation of mitogenesis. (iii) Finally, ChoP may bind to and enhance the function of the recently described S1P receptors (15, 16). Further experiments are required to distinguish between these possibilities. Also, the exact contribution by calcium to the potentiation of ChoP effects by ATP and S1P remains to be clarified.
An interesting question is how ChoP and ATP may affect the actions of other growth factors. Cuadrado et al. reported (6) that ChoP was also required for the actions of other growth factors such as platelet-derived growth factor and fibroblast growth factor. Although we could repeat their findings (6) that 5 mM hemicholinium-3, an inhibitor of choline kinase, inhibited the actions of these growth factors both on choline kinase activation and DNA synthesis, we also found that this drug has apparently nonspecific effects on DNA synthesis; e.g. this inhibitor was found to actually enhance the mitogenic effects of both insulin and ChoP. Thus, this agent may not be used for the study of the role of choline kinase in the regulation of mitogenesis. Most recently we expressed choline kinase in NIH 3T3 fibroblasts; these overexpressors are presently being used to clarify the role of ChoP in the mitogenic actions of various growth factors.
A potential problem of using the NIH 3T3 cell line is the apparent heterogeneity of cells in their response to growth regulatory agents. This prompted us to extend the studies on the mitogenic effects of ChoP, ATP, and insulin to the P-3T3 and N-3T3 subclones, which make up the major fraction of the NIH 3T3 line (9, 10). Evidently, growth regulation in the two subclones by the above agents is quite different, although the primary reason for the observed differences was the greatly reduced mitogenic activity of ChoP in the N-3T3 population. The fact that ATP restored the response of these cells to ChoP to a significant extent, particularly in the presence of insulin, indicated that using the clone 7 (original) NIH 3T3 cell population for these studies will not lead to misinterpretation of the data. In addition, N-3T3 cells may be used (since the base line of ChoP action is low) to identify other potentiating agents and to determine the exact mechanism(s) by which ChoP interacts with ATP, insulin, and S1P.
The major features of the combined mitogenic effects of ChoP, ATP, and
S1P are illustrated in the scheme shown in Fig. 12. Accordingly, ChoP, whose formation is stimulated by growth factors (59), carcinogens (60-62), and oncogenes (63, 64), is secreted from
cells by a process that is activated by extracellular ATP through a
purinergic P2-type cell surface receptor. The effect of
ChoP through an extracellular site is amplified by ATP and S1P by
mechanisms probably involving elevation of cytoplasmic calcium level
and activation of MAP kinase; presently, involvement of still another
mechanism(s) cannot be excluded. (We should note here that for
simplicity the interactions between the insulin receptor as well as the
ATP- and ChoP-induced mechanisms are not indicated in the scheme.) The
major effects of ChoP and ATP are mediated by PI 3-kinase and,
particularly, pp70s6k. A contribution by MAP kinase to the
mitogenic effects of ChoP and ATP appears to be minimal. While the
results strongly suggest that ChoP can be a potent mitogen when other
conditions are met, it is clear that much more work is needed to
clarify both the exact mechanisms involved and the physiological role
of these novel ChoP-dependent cell growth-regulatory
mechanisms.
We are grateful to Dr. Jean Y. J. Wang for
providing the P-3T3 and N-3T3 subclones, and to Dr. Masato Kasuga for
providing the plasmid SR-
p85.