(Received for publication, June 5, 1995)
From the
Elevation of intracellular cAMP by forskolin, 8-bromoadenosine
3`:5`-cyclic monophosphate, and prostaglandin E, in synergy
with insulin, stimulated DNA synthesis in quiescent Swiss 3T3 cells to
the same level achieved by platelet-derived growth factor (PDGF) or
bombesin. Both forskolin and 8-bromoadenosine 3`:5`-cyclic
monophosphate stimulated a significant increase in cell number which,
in the presence of insulin, reached the same levels achieved with PDGF.
Treatment with either PDGF or bombesin caused a marked and persistent
stimulation of p42
and p44
. In striking
contrast, no activation was seen with mitogenic combinations of cAMP as
shown by three different assays. Swiss 3T3 cells stably transfected
with a constitutively activated Gs
subunit were 100-fold more
sensitive to the mitogenic effects of forskolin but in this distinct
cellular model forskolin did not activate p42
. Swiss 3T3
cells stably transfected with interfering mutants of MEK-1 showed a 60%
decrease in PDGF-stimulated p42 MAPK activation, but there was no
inhibition of the mitogenic effect of forskolin in these cells.
Furthermore, the upstream kinases MEK-1/MEK-2 and p74
were not activated by mitogenic combinations of cAMP while PDGF
caused marked stimulation of their activity. Treatment of 3T3 cells
with forskolin attenuated PDGF-stimulated p74
and p42
activation but enhanced the mitogenic
effects of this agent. Mitogenic combinations of cAMP strongly
stimulated the phosphorylation and activation of p70
an
effect that was inhibited by rapamycin. This agent markedly inhibited
cAMP-stimulated DNA synthesis suggesting a critical role for
p70
in cAMP mitogenic signaling. These results
demonstrate that cAMP-induced mitogenesis can be dissociated from
activation of the mitogen-activated protein kinase cascade and that
this is not an obligatory point of convergence in mitogenic signaling
in Swiss 3T3 cells.
The mitogen-activated protein (MAP) ()kinases (ERKs)
are a family of highly conserved serine/threonine kinases that are
activated in response to a wide range of extracellular signals
including growth factors, hormones, and
neuropeptides(1, 2, 3) . The two best
characterized isoforms p42
(ERK-2) and p44
(ERK-1) (4) can be activated through both tyrosine kinase
receptors or G-protein-linked
receptors(1, 2, 3) . Once activated,
p42
and p44
phosphorylate an array of
cellular proteins including protein kinases such as
p90
(5) , transcription
factors(6, 7, 8) , and proteins involved in
the regulation of cell growth(9) . MAP kinases are themselves
activated by phosphorylation on specific threonine and tyrosine
residues by the dual-specificity MAP kinase kinase (or MEK) of which at
least two isoforms have been identified in mammalian
cells(10, 11, 12) . This kinase is itself
regulated by upstream kinases including the Raf family (13) and
MEK kinase(14) . Studies with dominant-negative and activating
mutants have provided evidence that this pathway can lead to the
stimulation of DNA synthesis(15, 16) . However, it is
unclear whether the activation of p42
and p44
is a point of convergence in the action of all signals that
promote DNA synthesis(17, 18, 19) .
The
cAMP-protein kinase pathway links a number of extracellular signals to
a range of cell functions including cell proliferation(20) .
Considerable evidence indicates that an increase in intracellular cAMP
can act as a mitogenic signal for Swiss 3T3 cells(21) . Agents
that promote cAMP production and accumulation, such as forskolin and
PGE, as well as permeable cAMP analogues, stimulate DNA
synthesis in 3T3 cells acting synergistically with insulin and other
factors(21, 22, 23, 24) . Cells
expressing a mutated cAMP-protein kinase regulatory subunit show
markedly reduced cAMP-protein kinase activation and mitogenesis in
response to agents that elevate intracellular cAMP (25) .
Conversely, Swiss 3T3 cells expressing a constitutively active Gs
subunit are highly sensitive to the mitogenic effects of cAMP elevating
agents(26) . Increases in cAMP also lead to early intracellular
events associated with cell proliferation including an increase in the
expression of the proto-oncogene c-myc. (27) .
However, the exact relationship between the mitogenic effect of cAMP
and activation of the MAP kinase cascade is as yet not defined.
Here
we report that the mitogenic effects of cAMP are not associated with
detectable activation of p42 and p44
,
MEK-1/-2, or of p74
. Interfering mutants of
MEK-1 stably transfected into Swiss 3T3 cells significantly inhibited
PDGF-stimulated p42
activation but did not inhibit
cAMP-induced mitogenesis. Further dissociation of mitogenesis from the
MAPK cascade is demonstrated by the finding that elevating
intracellular cAMP inhibits PDGF-stimulated p74
and p42
activation but enhances
PDGF-stimulated DNA synthesis. Mitogenic combinations of cAMP strongly
stimulated the phosphorylation and activation of p70
, an
effect that was inhibited by rapamycin. This agent markedly inhibited
cAMP-stimulated DNA synthesis identifying this as a distinct pathway in
cAMP mitogenic signaling.
Figure 1:
cAMP stimulates DNA synthesis in Swiss
3T3 cell but does not activate p42 and
p44
. A, confluent and quiescent cultures of
Swiss 3T3 cells were washed and incubated at 37 °C in 2 ml of
DMEM/Waymouth's medium containing 1 µCi/ml of
[
H]thymidine and various factors: 10 µM forskolin with 50 µM IBMX (FSK), 2.5 mM 8-BrcA (BrcA), 10 ng/ml bombesin (Bom), 10 ng/ml
PDGF, or no addition(-), either in the absence or presence of 1
µg/ml of insulin (Ins). After 40 h, DNA synthesis was
assessed by measuring the [
H]thymidine
incorporated into acid-precipitable material. Results are expressed as
a percentage of the incorporation induced by 10% FBS, and data are
shown as mean ± S.E. (n = 14). B, upper panel, parallel, confluent, and quiescent cultures of
Swiss 3T3 cells were washed and treated for 5 min at 37 °C with
factors as above, lysed in SDS sample buffer, and analyzed by Western
blotting with anti-p42
polyclonal antibody. The
positions of non-phosphorylated p42
and the slower
migrating phosphorylated form pp42
are indicated. B, lower panel, confluent and quiescent cultures of
Swiss 3T3 cells were washed and treated for 5 min at 37 °C with
factors as above and lysed in sample buffer. Samples were analyzed
using in situ phosphorylation of MBP by renaturable kinases
following SDS-PAGE as described under ``Experimental
Procedures.'' The positions of p42
and p44
are indicated. C, confluent and quiescent cultures of
Swiss 3T3 cells were washed and treated for 5 min at 37 °C with
factors as above, lysed in lysis buffer, immunoprecipitated with
anti-p42
polyclonal antibody, and the immune complexes
analyzed in an immune complex kinase assay using MBP peptide as a
substrate (see ``Experimental Procedures''). Results are
expressed as cpm/1.5
10
cells, and the data are
shown as the mean ± S.E. for four independent experiments each
performed in duplicate. The specific activity of
[
-
P]ATP used was 900-1200
cpm/pmol.
To assess whether treatment of Swiss 3T3 cells with cAMP-elevating agents resulted in cell proliferation, cultures of 3T3 cells in conditioned medium were treated for 72 h with the various factors and cell number determined. As shown in Table 1addition of either forskolin or 8-BrcA alone both caused a statistically significant increase in cell number compared to control cultures. In the presence of insulin, the stimulatory effect of forskolin was comparable to that achieved with PDGF.
To examine the
effects of these mitogenic factors upon the activity of p42 and p44
, cell lysates from parallel cultures were
analyzed by Western blotting using specific polyclonal antibodies to
these proteins. Activation was determined by the appearance of slower
migrating forms which results from the phosphorylation of specific
threonine and tyrosine residues in these kinases(29) .
Treatment of cells with PDGF or bombesin, either alone or in
combination with insulin, stimulated p42
as judged by
the mobility shift (Fig. 1B, upper panel). In
contrast, no slower migrating form was seen with the mitogenic
combinations of either forskolin or 8-BrcA with insulin or with insulin
alone. Similar findings were obtained with p44
(data not
shown).
The activation of p42 and p44
in response to mitogens was further examined using in situ phosphorylation of MBP by renaturable kinases following SDS-PAGE. Fig. 1B, lower panel, shows that bombesin and
PDGF stimulated MBP kinases of molecular masses 42 and 44 kDa
corresponding to p42
and p44
. No
activation of these MBP kinases was seen in response to the mitogenic
combinations of either forskolin or 8-BrcA with insulin or with insulin
alone. No additional MBP kinases of higher or lower molecular weight
were activated by increasing intracellular cAMP (data not shown). This
has been further corroborated by immune complex kinase assays using
anti-p42
immunoprecipitates from cells treated with each
of the mitogens. Fig. 1C shows that while PDGF and
bombesin, either alone or in combination with insulin, stimulated
p42
activity, the cAMP elevating agents in combination
with insulin did not induce a significant increase in p42
activation. The results depicted in Fig. 1suggest a
striking dissociation of p42
and p44
activation (as assessed by three distinct assay techniques) from
the mitogenic effects of cAMP, a finding that is in marked contrast
with the activation stimulated by PDGF and bombesin.
Figure 2:
cAMP does not activate p42 and p44
over short and long time courses. A, confluent and quiescent cultures of Swiss 3T3 cells were
washed and treated with either 10 ng/ml bombesin (Bom) or 10
µM forskolin and 50 µM IBMX with 1 µg/ml
of insulin (FSK) for the times indicated, lysed in sample
buffer, and analyzed by Western blotting with anti-p42
polyclonal antibody. The positions of non-phosphorylated
(p42
) and the slower migrating phosphorylated form
pp42
are indicated. B, confluent and quiescent
cultures of Swiss 3T3 cells were washed and treated with either 10
ng/ml bombesin (
), 10 µM forskolin and 50
µM IBMX with 1 µg/ml of insulin (
), or control
medium (serum-free DMEM) (
) for the times indicated lysed in
lysis buffer, immunoprecipitated with anti-p42
polyclonal antibody, and the immune complexes analyzed in an
immune complex kinase assay using MBP peptide as a substrate (see
``Experimental Procedures''). Results are the means of
duplicates and are expressed as percent of bombesin-stimulated
activation (1400-1600 cpm/1.5
10
cells at 5
min) and are representative of three independent experiments. The
specific activity of [
-
P]ATP used was
900-1200 cpm/pmol.
To further assess the kinetics of p42 activation,
immune complex kinase assays were performed over a time course of up to
120 min. As shown in Fig. 2B, bombesin stimulated a
persistent activation in p42
activity beyond 120 min
while forskolin and insulin did not induce a significant increase in
activity over the same time period compared to untreated control cells.
Figure 3:
PGE is a potent mitogen but
does not activate p42
. A, confluent and
quiescent cultures of Swiss 3T3 cells were washed and incubated at 37
°C in 2 ml of DMEM/Waymouth's medium containing 1 µCi/ml
of [
H]thymidine and various factors: 1 µg/ml
of insulin (Ins), 50 ng/ml PGE
with 25 µM IBMX and 1 µg/ml of insulin (PGE
), 10
ng/ml bombesin (Bom) or no addition(-). After 40 h, DNA
synthesis was assessed by measuring the
[
H]thymidine incorporated into acid-precipitable
material. Results are expressed as a percentage of the incorporation
induced by 10% FBS, and data are shown as mean ± S.E. (n = 6). B, confluent and quiescent cultures of Swiss
3T3 cells were washed and treated with PGE
with 25
µM IBMX and 1 µg/ml of insulin (PGE
) for
the times indicated at 37 °C, lysed in sample buffer, and analyzed
by Western blotting with anti-p42
polyclonal antibody.
Cells stimulated with 10 ng/ml bombesin for 5 min (Bom) were
used as a positive control. The positions of non-phosphorylated
p42
and the slower migrating phosphorylated form
pp42
are indicated. C, confluent and quiescent
cultures of Swiss 3T3 cells were washed and treated for 5 min in DMEM
with factors as in A above, lysed in lysis buffer,
immunoprecipitated with anti-p42
polyclonal antibody,
and the immune complexes analyzed in an immune complex kinase assay
using MBP peptide as a substrate (see ``Experimental
Procedures''). Results are expressed as cpm/1.5
10
cells, and the data are shown as the mean ± S.E. for three
independent experiments each performed in duplicate. The specific
activity of [
-
P]ATP used was 900-1200
cpm/pmol.
Figure 4:
Effect of forskolin on Swiss 3T3 cells
transfected with a constitutively activated Gs subunit. A, confluent and quiescent cultures of either wild type Swiss
3T3 cells (3T3) or Swiss 3T3 cells transfected with a
constitutively activated Gs
subunit (Gs
(Q227L
s) 3T3) were washed and incubated at 37 °C in 2 ml of
DMEM/Waymouth's medium containing 1 µCi/ml of
[
H]thymidine and various factors: 1 µg/ml of
insulin (Ins), 10 µM forskolin with 1 µg/ml
of insulin (FSK 10) for the wild type cells, and 0.1
µM forskolin with 1 µg/ml of insulin (FSK
0.1) for the Gs
(Q227L
s) 3T3 cells, or no addition
(-). After 40 h, DNA synthesis was assessed by measuring the
[
H]thymidine incorporated into acid-precipitable
material. Results are expressed as a percentage of the incorporation
induced by 10% FBS, and data are shown as mean ± S.E. (n = 8). B, confluent and quiescent cultures of
Gs
(Q227L
s) 3T3 cells were washed and treated with 10 ng/ml
bombesin (Bom), 0.1 µM forskolin with 1 µg/ml
of insulin (FSK 0.1), 10 µM forskolin with 1
µg/ml of insulin (FSK 10) or vehicle(-) for 5 min at
37 °C, lysed in sample buffer, and analyzed by Western blotting
with anti-p42
polyclonal antibody. The positions of
non-phosphorylated p42
and the slower migrating
phosphorylated form pp42
are indicated. C,
confluent and quiescent cultures of Gs
(Q227L
s) 3T3 cells were
washed and treated for 5 min with 0.1 µM forskolin with 1
µg/ml of insulin (FSK 0.1), 10 ng/ml bombesin (Bom), or no addition(-), lysed in lysis buffer,
immunoprecipitated with anti-p42
polyclonal antibody,
and the immune complexes analyzed in an immune complex kinase assay
using MBP peptide as a substrate (see ``Experimental
procedures''). Results are expressed as cpm/1.5
10
cells, and the data are shown are the means ± S.E. for two
independent experiments each performed in duplicate. The specific
activity of [
-
P]ATP used was 900-1200
cpm/pmol.
Figure 5:
Interfering mutants of MEK-1 significantly
inhibit PDGF-stimulated p42 activation but do not
inhibit cAMP-induced mitogenesis. A, confluent and quiescent
cultures of untransfected Swiss 3T3 cells (3T3) or those
overexpressing wild type (WT), or mutant MEK-1 with alanine
for serine substitutions (Ala
and Ala
) were lysed in sample buffer and normalized
for cell number and analyzed by Western blotting with anti-MEK-1
monoclonal antibody. Comparable levels of overexpression of MEK-1 (a
6-fold increase over untransfected cells as assessed by scanning
densitometry of autoradiographs) are demonstrated in the clones used in
the subsequent experiments. B, confluent and quiescent
cultures of untransfected and Swiss 3T3 cells overexpressing WT,
Ala
, and Ala
MEK-1 were washed and treated
for 5 min at 37 °C with 10 ng/ml PDGF, lysed in lysis buffer,
immunoprecipitated with anti-p42 MAPK polyclonal antibody, and the
immune complexes analyzed in an immune complex kinase assay using MBP
peptide as a substrate (see ``Experimental Procedures'').
Results are expressed as percent of maximum PDGF-stimulated activation
(2000-2500 cpm/1.5
10
cells at 5 min), and
the data are shown as the mean ± S.E. for three independent
experiments each performed in duplicate. The specific activity of
[
-
P]ATP used was 900-1200 cpm/pmol. C, confluent and quiescent cultures of untransfected and Swiss
3T3 cells overexpressing WT, Ala
, and Ala
MEK-1 were washed and incubated at 37 °C in 2 ml of
DMEM/Waymouth's medium containing 1 µCi/ml of
[
H]thymidine and 10 µM forskolin
with 50 µM IBMX and 1 µg/ml of insulin (FSK).
After 40 h, DNA synthesis was assessed by measuring the
[
H]thymidine incorporated into acid-precipitable
material. Results are expressed as a percentage of the incorporation
induced by 10% FBS, and data are shown as mean ± S.E. (n = 8).
Figure 6:
Mitogenic combinations of cAMP do not
activate MEK-1 and MEK-2 or p74. A,
confluent and quiescent cultures of Swiss 3T3 cells were washed and
treated for 5 min with 10 µM forskolin and 50 µM IBMX with 1 µg/ml of insulin (FSK), 10 ng/ml bombesin (Bom), 10 ng/ml PDGF, or no addition(-), lysed in lysis
buffer, immunoprecipitated with anti-MEK-1/MEK-2 polyclonal antibody,
and the immune complexes analyzed in a two-step immune complex kinase
assay as described under ``Experimental Procedures.'' Results
are expressed as percent of maximum PDGF-stimulated activation
(19000-25000 cpm/1.5
10
cells at 5 min), and
the data are shown as the mean ± S.E. for two independent
experiments each performed in triplicate. The specific activity of
[
-
P]ATP used was 900-1200 cpm/pmol. B, confluent and quiescent cultures of Swiss 3T3 cells were
washed and treated with either 10 ng/ml PDGF (
), 10 µM forskolin and 50 µM IBMX with 1 µg/ml of insulin
(
), or with control medium (serum-free DMEM) (
) for the
times indicated, lysed in lysis buffer, immunoprecipitated with
anti-p74
polyclonal antibody, and the immune
complexes analyzed in a two-step immune complex kinase assay as
described in ``Experimental Procedures.'' Results are the
means of duplicates and are expressed as percent of maximum
PDGF-stimulated activation (5000-6000 cpm/1.5
10
cells at 5 min) and are representative of three independent
experiments. The specific activity of
[
-
P]ATP used was 900-1200
cpm/pmol.
As cAMP is a mitogen in Swiss 3T3 cells, rather than growth
inhibitory as it is in Rat-1 cells, we examined the effect of forskolin
upon PDGF stimulated p74 and p42
activation. Preincubation with forskolin abolished
PDGF-stimulated p42
activation as shown in the mobility
shift assay (Fig. 7A). This effect appeared to be
mediated at the level of p74
as
PDGF-stimulated activation of this kinase was markedly inhibited by
cAMP (Fig. 7B). However, PDGF-stimulated DNA synthesis
in the presence of cAMP elevating agents was significantly enhanced (p <0.001) (Fig. 7C). In other experiments,
we found that forskolin augmented [
H]thymidine
incorporation induced by PDGF at 2.5 and 5 ng/ml by 178 and 205%,
respectively (data not shown). These findings again illustrate
dissociation of activation of the MAP kinase cascade from cAMP-induced
mitogenesis.
Figure 7:
cAMP pretreatment attenuates
PDGF-stimulated activation of p74 and
p42
but enhances its mitogenic effects. A,
confluent and quiescent cultures of Swiss 3T3 cells were washed and
treated for 5 min at 37 °C with 10 ng/ml PDGF either with (+)
or without(-) 5 min of pretreatment with 50 µM forskolin and 50 µM IBMX (FSK) or with
vehicle(-), lysed in sample buffer, and analyzed by Western
blotting with anti-p42
polyclonal antibody. The
positions of non-phosphorylated p42
and the slower
migrating phosphorylated form pp42
are indicated. B, confluent and quiescent cultures of Swiss 3T3 cells were
washed and treated for 5 min at 37 °C with 10 ng/ml PDGF, 10 ng/ml
PDGF with 5 min pretreatment with 50 µM forskolin and 50
µM IBMX (PDGF + FSK), or with
vehicle(-), lysed in lysis buffer, immunoprecipitated with
anti-p74
polyclonal antibody, and the immune
complexes analyzed in a two-step immune complex kinase assay as
described under ``Experimental Procedures.'' Results are
expressed as percent of maximum PDGF-stimulated activation
(5000-6000 cpm/1.5
10
cells at 5 min), and
the data are shown as the mean ± S.E. for three independent
experiments each performed in duplicate. The specific activity of
[
-
P]ATP used was 900-1200 cpm/pmol. C, confluent and quiescent cultures of Swiss 3T3 cells were
washed and incubated at 37 °C in 2 ml of DMEM/Waymouth's
medium containing 1 µCi/ml of [
H]thymidine
and either 10 ng/ml PDGF,10 ng/ml PDGF with 50 µM
forskolin and 50 µM IBMX (PDGF + FSK) or no
addition(-). After 40 h, DNA synthesis was assessed by measuring
the [
-H]thymidine incorporated into
acid-precipitable material. Results are expressed as a percentage of
the incorporation induced by 10% FBS, and data are shown as mean
± S.E. (n = 10). The combination of PDGF with
forskolin and IBMX gave a significantly greater stimulation of DNA
synthesis compared to PDGF alone (p < 0.001,
Student's t test for unpaired
data).
As shown in Table 2, treatment
of Swiss 3T3 cells with either forskolin or insulin stimulated an
increase in p70 activity measured in an immune complex
kinase assay by 2.5- and 6-fold, respectively. The mitogenic
combination of forskolin and insulin induced a further stimulation of
p70
activity. This stimulatory effect was completely
inhibited by rapamycin at 20 ng/ml (Table 2). Fig. 8, upper panel, shows in a mobility shift assay that forskolin in
combination with insulin stimulated the phosphorylation of p70
which accompanies the activation of this kinase. The mobility
shift induced by these agents was comparable to that induced by PDGF
which is presented for comparison (Fig. 8). In parallel
experiments both forskolin and 8-BrcA alone also induced shift of
p70
(results not shown). The phosphorylation of
p70
induced by the mitogenic combination of forskolin
with insulin was strongly inhibited by rapamycin. The striking finding
shown in Fig. 8, lower panel, is that rapamycin
markedly inhibited cAMP-stimulated DNA synthesis. The results presented
in Fig. 8thus identify one of the pathways utilized by cAMP and
insulin to induce DNA synthesis that is distinct from the MAP kinase
cascade.
Figure 8:
Mitogenic combinations of cAMP stimulate
the phosphorylation of p70 and inhibition of p70
by rapamycin inhibits cAMP-induced mitogenesis. Upper
panel, confluent and quiescent cultures of Swiss 3T3 cells were
washed and treated for 15 min at 37 °C with 10 µM forskolin with 50 µM IBMX and 1 µg/ml of insulin (FSK), 10 ng/ml PDGF, or with vehicle alone(-) with or
without 30 min of pretreatment with rapamycin 20 ng/ml. Lysates were
analyzed by Western blotting as described under ``Experimental
Procedures.'' The mobility shift upon phosphorylation and
activation of the
II isoform of p70
is shown with
the phosphorylated form of the kinase being indicated by an arrow. Lower panel, confluent and quiescent cultures
of Swiss 3T3 cells were washed and incubated at 37 °C in 2 ml of
DMEM/Waymouth's medium containing 1 µCi/ml of
[
H]thymidine and factors as above. After 40 h,
DNA synthesis was assessed by measuring the
[
H]thymidine incorporated into acid-precipitable
material. Results are expressed as a percentage of the incorporation
induced by 10% FBS, and data are shown as mean ± S.E. (n = 8).
The reinitiation of DNA synthesis in G-arrested
cells can be induced by multiple signaling pathways that act in a
combinatorial and synergistic
fashion(21, 43, 44) . In the present study we
utilized PDGF, bombesin, and cAMP in combination with insulin to
promote maximum levels of DNA synthesis in Swiss 3T3 cells. Although
these mitogenic signals must converge prior to DNA replication, the
precise point of convergence in G
remains unknown. The
redundancy in signaling pathways prior to the point of convergence
implies that some events may be sufficient to stimulate DNA synthesis
but not neccesarily obligatory for the action of all mitogens.
p42
and p44
are activated by a number of
mitogenic signaling pathways linked to both tyrosine kinase and
G-protein-linked receptors(1, 2, 3) . These
include the Raf and MEK kinase pathways which transduce the signals
from such effectors as p21
and PKC. p42
and p44
have been reported to phosphorylate an
array of proteins involved in the mitogenic response including
p90
(5) , the proto-oncogene products
c-myc(7) and c-jun(8) , and the
transcription factor TCF 62(6) . Sustained activation of
p42
and p44
, therefore, may be an
obligatory step in the action of all mitogens leading to DNA synthesis
as, in fact, has been proposed(45) . However, several recent
observations suggest that activation of p42
or
p44
may not be an obligatory point of convergence in the
action of all mitogenic signals. Interleukin-4 stimulates proliferation
of two cell lines of T-lymphocyte and myeloid origin but not does
activate MAP kinase(18) . Additionally, thyroid-stimulating
hormone stimulates mitogenesis in thyrocytes but does not stimulate
tyrosine phosphorylation of MAP kinase (17, but see also 46). In Swiss
3T3 cells, activin, a member of the transforming growth factor
family of cytokines, is mitogenic but appears not to stimulate MAP
kinase activation(19) . These findings suggest but do not prove
that p42
and p44
activation is not
obligatory for DNA synthesis because the sensitivity of the assays used
has been questioned (47) and none of the studies examined the
effects of interfering mutants that block MAP kinase activation in
vivo.
The results presented in this study demonstrate that the
potent mitogenic effects of cAMP are not mediated via the MAP kinase
cascade in Swiss 3T3 cells. Agents that elevate intracellular cAMP by
distinct mechanisms are able to stimulate DNA synthesis to a level
comparable to that seen with PDGF and bombesin. Additionally,
cAMP-elevating agents either alone or in combination with insulin
induce a significant increase in cell number demonstrating that these
agents also stimulate progression through later stages of the cell
cycle. However, using three separate assays we have shown that cAMP
fails to detectably activate either p42 or
p44
. This is in marked contrast to the effects of PDGF
and bombesin which gave prolonged activation of these kinases.
Interfering mutants of MEK-1 stably transfected into Swiss 3T3 also
provide further convincing evidence that MAP kinase activation is not
involved in cAMP-induced mitogenesis. In these cells PDGF-stimulated
MAP kinase activation was significantly attenuated but the mitogenic
effect of forskolin was uninhibited. In line with this conclusion, cAMP
does not cause a significant induction of c-fos(27) ,
the expression of which is regulated by the MAP kinase cascade through
phosphorylation of TCF 62(48) . Swiss 3T3 cells stably
transfected with a constitutively activated Gs
subunit are highly
sensitive to the mitogenic effects of forskolin, and yet in this
distinct cellular model again we did not detect p42
activation in response to this cAMP elevating agent. Furthermore,
we have shown that cAMP fails to stimulate the upstream kinases in the
cascade namely p74
(see below) and
MEK-1/MEK-2. In contrast, under identical experimental conditions we
have confirmed that PDGF and bombesin potently stimulate p42
and p44
together with the upstream kinase MEK-1/-2
in Swiss 3T3 cells. Thus in this study we have demonstrated that cAMP
does not utilize proteins at three levels of the MAP kinase cascade
dissociating its mitogenic effects from a persistent activation of this
pathway.
The serine/threonine protein kinase p74 has been shown to play a central role in the mitogenic
response of cells to growth factors and many oncogenes(49) . It
associates with activated Ras and stimulates the downstream elements of
the MAP kinase cascade(50) . However, our results also provide
evidence that p74
activation is not an
obligatory step in cAMP mitogenic signal transduction. Mitogenic
combinations of cAMP do not induce a significant increase in
p74
activity. Indeed, an increase in cAMP
strikingly inhibited the activation of p74
and
p42
by PDGF but significantly increased the mitogenic
effect of PDGF. Our results therefore demonstrate, for the first time,
that cAMP can stimulate cell proliferation and inhibit the MAP kinase
cascade in the same cell type providing further evidence for a
dissociation between mitogenesis and activation of this pathway.
It
is now clear that cAMP mitogenic signaling is not confined to Swiss 3T3
cells. Agents which elevate intracellular cAMP are mitogenic for number
of cell types including mammary, keratinocyte, and kidney epithelial
cells(51) . Additionally, growth hormone-releasing factor,
which stimulates cAMP accumulation, is mitogenic for the rat anterior
pituitary somatotroph(51) . Further insight into the importance
of the cAMP pathway in cell proliferation has come with the
identification of constitutively activated Gs subunits in a
variety of tumors(52) . These mutations, which result in a
persistently elevated intracellular cAMP, are potentially oncogenic and
demonstrate that cAMP may play a role in cellular transformation.
Interestingly, our results with Swiss 3T3 cells stably transfected with
a constitutively activated Gs
subunit indicate that cAMP can
initiate a mitogenic response that is not mediated via the MAP kinase
cascade in these cells.
Many growth factors activate a parallel but
distinct signaling pathway leading to the phosphorylation and
activation of p70 which rapidly phosphorylates the S6
protein of 40 S ribosomal subunit (43) . Inhibition of the
activation of this enzyme with the immunosuppressant rapamycin or with
neutralizing antibodies demonstrates that p70
plays a
role in serum-stimulated mitogenesis(33, 53) .
Interestingly, it has been recently reported that p70
phosphorylates CREM, which is also a target for cAMP-protein
kinase, in response to mitogenic factors leading to a strong increase
in transcriptional activation(54) . This identifies a point of
convergence between the p70
and cAMP-protein kinase
pathways that is distinct from the nuclear targets of the MAP kinase
cascade. Our results show that cAMP in combination with insulin
stimulates the activity and phosphorylation of p70
as
demonstrated by immune complex kinase and mobility shift assays.
Rapamycin completely prevented the increase in activity and
phosphorylation induced by cAMP. Crucially, we show here for the first
time that rapamycin markedly attenuated the mitogenic effect of cAMP in
combination with insulin. Thus our results identify p70
,
as opposed to MAP kinase, MEK, and Raf-1, as an important element in
cAMP-induced mitogenic signaling pathways.
In conclusion, our
results demonstrate that cAMP, a potent mitogen for Swiss 3T3 cells,
does not induce a significant and persistent activation of p42 and p44
and fails to stimulate the upstream
kinases of the cascade namely p74
and MEK1/2.
These findings, dissociating the mitogenic effects of cAMP from
activation of the MAP kinase cascade, support the hypothesis that this
kinase cascade is one of the parallel pathways that can lead to DNA
synthesis rather than an obligatory point of convergence in mitogenic
signaling.