(Received for publication, July 8, 1996, and in revised form, September 12, 1996)
From the Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
In a previous study, we demonstrated that
parathyroid hormone (PTH) inhibits mitogen-activated protein (MAP)
kinase activation in osteosarcoma cells via a protein kinase
A-dependent pathway. Here, we show that PTH can induce a
transient activation of MAP kinase as well. This was observed in both
Chinese hamster ovary R15 cells stably expressing high levels of rat
PTH/PTH-related peptide receptor and parietal yolk sac carcinoma cells
expressing the receptor endogenously. PTH was a strong activator of
adenylate cyclase and phospholipase C in Chinese hamster ovary R15
cells. PTH-induced MAP kinase activation did not depend on activation of Gi, phorbol ester-sensitive protein kinase C, elevated
intracellular calcium levels, or release of G subunits. It could,
however, be mimicked by addition of forskolin or 8-bromo-cAMP to these cells. Prolonged treatment with forskolin caused sustained protein kinase A activity, whereas MAP kinase activity returned to basal levels. Subsequent treatment with PTH or 8-bromo-cAMP did not result in
MAP kinase activation, whereas phorbol ester- or insulin-induced MAP
kinase activation was unaffected. Finally, expression of a dominant
negative form of Ras (RasAsn-17), which completely blocked
insulin-induced MAP kinase activation, did not affect activation by PTH
or cAMP. In conclusion, PTH regulates MAP kinase activity in a cell
type-specific fashion. The activation of MAP kinase by PTH is mediated
by cAMP and independent of Ras.
Mitogen activated protein (MAP)1 kinases are protein serine and threonine kinases that play an important role in the regulation of cell growth and differentiation (1-3). The activity of MAP kinase is under the control of external stimuli that mediate their effects by binding to cell surface receptors. Protein tyrosine kinase receptors transduce the signal by autophosphorylation of tyrosine residues, allowing the receptor to interact with Src homology 2 domain-containing proteins, such as Grb2, which will recruit son of sevenless, resulting in the activation of Ras. This will cause the successive activation of Raf-1, MAP kinase kinase (MEK), and MAP kinase (4).
G protein-coupled receptors (GPCRs) regulate MAP kinase activity,
depending on the identity of the G protein, the receptor, and the cell
type involved. Gi-coupled receptors, such as the M2
muscarine acetylcholine receptor, the 2-adrenergic
receptor, or the receptors for lysophosphatidic acid (LPA) or thrombin, stimulate MAP kinase in a Ras-dependent manner (5-7).
Recent reports have shown that MAP kinase activation through
Gi involves the release of G
subunits (8-11), which
via an as yet unidentified tyrosine kinase induce the phosphorylation
of Shc, leading to the formation of a Shc-Grb2-son of sevenless complex
(12, 13) and activation of Ras.
For receptors coupled to Gq, such as the M1 acetylcholine
receptor, the 1-adrenergic receptor, or the bombesin
receptor, both
subunit-dependent (10) and
subunit-independent (8, 11) activation of MAP kinase has been reported.
The
subunit-induced activation is mediated by Ras (10), whereas
the Gq
-induced activation is mediated by phorbol
ester-sensitive protein kinase C (PKC) in a Ras-independent manner
(11).
Gs-coupled receptors, such as the -adrenergic receptor
or the pituitary adenylate cyclase-activating polypeptide receptor, can
either trigger or inhibit MAP kinase activation (14-16). This is cell
type-dependent and can in most cases be mimicked by
addition of cell-permeable cAMP analogues. With respect to the
inhibition of MAP kinase activation, it was demonstrated that
activation of the cAMP-dependent protein kinase A (PKA)
interfered with Ras-mediated activation of MAP kinase at the level of
Raf-1 (17). However, the mechanism behind the cAMP-mediated activation
of MAP kinase, as in PC12 cells, is largely unclear (16, 18).
PTH and PTHrP bind to a common receptor, which has been shown to couple
to at least two signal transduction systems: (i) a Gs-mediated increase in cAMP, leading to activation of PKA
(19-21); and (ii) a Gq-mediated activation of PLC-,
leading to increases in intracellular inositol triphosphate and calcium
levels and activation of PKC (19, 21-23). The identity of downstream
effectors and their role in cellular responses to PTH and PTHrP are
unclear. We have recently demonstrated that PTH inhibits growth
factor-induced MAP kinase activation in osteosarcoma cells via a
pathway that is dependent on PKA (24).
Here we show for two cell types that triggering of the PTH/PTHrP
receptor can also lead to activation of MAP kinase. This activation was
not dependent on three well established Gq-mediated events,
i.e.: (i) elevation of intracellular calcium levels, (ii) activation of PKC, and (iii) the release of G subunits. We
provide evidence that the effect is mediated by elevation of
intracellular cAMP levels and occurs in a Ras-independent manner.
Rat PTH(1-34) was purchased from Peninsula
Laboratories Europe (St. Helens, United Kingdom). 8-Bromo-cAMP and
thapsigargin were obtained from Biomol (Plymouth Meeting, PA).
Thrombin, insulin, LPA, isoproterenol,
12-O-tetradecanoylphorbol-13-acetate (TPA), forskolin,
pertussis toxin (PTX), and the peptides Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), myelin basic protein, and protein kinase inhibitor were
from Sigma. [32P]ATP and ECL were
purchased from Amersham Corp. Indo-1 acetoxymethyl ester was obtained
from Molecular Probes (Eugene, OR). Polyclonal antibodies against p42
MAP kinase were kindly provided by Drs. J. L. Bos and B. M. T.
Burgering (Utrecht University, Utrecht, The Netherlands).
CHO-R15, CHO-2, and
CHO-K1 cells were grown in Dulbecco's modified Eagle's medium
supplemented with 7.5% fetal calf serum (FCS). PYS-2 cells were grown
in medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's
medium and Ham's F12 containing 7.5% FCS. Transient transfections
were performed using the calcium phosphate precipitation method. One
day prior to transfection, the CHO-R15 cells were plated at a density
of 8 × 103 cells/cm2 in six-well tissue
culture clusters. The following day they were cotransfected with
plasmid DNA encoding p44 HA-MAP kinase (2 µg/well), G
subunit of
retinal transducin (G
t) (1 µg/well), or
RasAsn-17 (3 µg/well). Puc-Rous sarcoma virus plasmid was
added to bring the total amount of plasmid DNA to 10 µg/well.
Nearly confluent CHO-R15 monolayers, attached to rectangular glass coverslips, were incubated in Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum for 18 h. The cells were loaded with indo-1 by exposing them to 10 µM indo-1 ester for 40 min at 37 °C. [Ca2+]-dependent fluorescence was recorded at an excitation wavelength of 355 nm and an emission of 405 nm (25). Since we were not able to perform proper calibration of the calcium responses in CHO-R15 cells, we related the calcium response to PTH(1-34) to that to thrombin (1 arbitrary unit).
Activation and Phosphorylation of MAP KinaseThe CHO and PYS-2 cells were plated in concentrations of, respectively, 2.5 × 104 and 1 × 104 cells/cm2 in six-well tissue culture clusters and grown for 24 h. The cells were incubated in medium containing 0.5% FCS for 18 h and subsequently treated with agents as indicated. MAP kinase phosphorylation was measured by Western blotting with anti-p42 MAP kinase antibodies as described previously (24). Phosphorylated p42 MAP kinase is detected as a band with reduced mobility compared with unphosphorylated p42 MAP kinase (26). Experiments were repeated at least three times, and representative results are shown.
For determination of MAP kinase activity, endogenous p42 MAP kinase and epitope-tagged p44 HA-MAP kinase were immunoprecipitated with protein A-Sepharose beads coupled to, respectively, anti-p42 MAP kinase antibodies and monoclonal antibody 12CA5, as described previously (24, 27). After the kinase reaction with myelin basic protein as a substrate, the reaction mix was subjected to SDS-polyacrylamide gel electrophoresis. Phosphorylation of myelin basic protein was measured using a PhosphorImager and ImageQuant software (Molecular Dynamics).
PKA ActivationCHO-R15 cells were plated at a concentration of 2.5 × 104 cells/cm2 in 96-well tissue culture clusters and grown for 24 h. The cells were incubated in medium containing 0.5% FCS for 18 h and subsequently treated with agents as indicated.
Activation of PKA was measured as described previously (24, 28).
Briefly, digitonin-permeabilized cells were incubated with a salt
solution containing [-
32P]ATP and Kemptide as a
substrate, with or without protein kinase inhibitor as an inhibitory
peptide. After a 10-min incubation, the reaction was stopped with 25%
trichloroacetic acid, and the trichloroacetic acid-soluble material was
spotted on phosphocellulose filters. The difference in radioactivity
incorporated in the filters between samples treated with protein kinase
inhibitor- versus non-protein kinase inhibitor-treated
samples was defined as PKA activity.
Whereas in most
cell types the PTH/PTHrP receptor is a potent activator of
Gs, the activation of Gq is suggested to be
dependent on receptor density (29). To investigate the effect of PTH on MAP kinase activity in a situation in which the receptor couples strongly to both Gs and Gq (21, 29), we used
Chinese hamster ovary cells stably transfected with the rat PTH/PTHrP
receptor (CHO-R15). Binding studies with a radioiodinated PTH analogue, PTH(1-34), revealed that these cells express approximately 300,000 PTH/PTHrP receptors per cell.2 Activation
of MAP kinase was detected using a gel mobility shift assay (26).
Treatment of the cells with 107 M PTH(1-34)
induced a transient phosphorylation of MAP kinase, which was maximal
after 5-10 min and returned to a basal level within 60 min (Fig.
1A). PTH(1-34) induced MAP kinase
phosphorylation in a dose-dependent manner, starting at
10
11 M (Fig. 1B).
PTH-induced MAP Kinase Activation Is Not Dependent on Gi, G
Well established pathways
used by GPCRs in the activation of MAP kinase involve Gi or
PKC (5-7, 11, 30, 31). We tested their involvement in the PTH-induced
MAP kinase activation by a prolonged treatment of the cells with PTX
and TPA to respectively inhibit Gi and down-modulate PKC.
As expected, PTX inhibited the thrombin-induced MAP kinase activation
by approximately 80%, whereas prolonged TPA treatment completely
inhibited the TPA-induced MAP kinase activation. Neither of the
treatments affected the activation of MAP kinase by PTH (data not
shown). Combined treatment with PTX and TPA completely inhibited the
activation of MAP kinase by both thrombin and TPA but had no effect on
the activation of MAP kinase by PTH(1-34) (Fig. 2).
Thus it appears that the PTH-induced MAP kinase activation is not
dependent on Gi or TPA-sensitive PKC.
Stimulation of CHO-R15 cells with PTH(1-34) induced a transient
increase in intracellular calcium, as shown in Fig.
3A. Several reports demonstrate that an
increase in intracellular calcium can be sufficient to activate MAP
kinase (32-35). We first examined whether PTH mobilizes calcium from
an intracellular or extracellular source by incubating the cells with
thapsigargin. This depletes calcium from intracellular stores (36). As
is demonstrated in Fig. 3A for FCS, the rapid component, due
to release of calcium from intracellular stores, was completely
inhibited by a 40-min thapsigargin pretreatment, whereas the slower
component, due to calcium influx, was not. The calcium response to
PTH(1-34) was completely inhibited by thapsigargin, suggesting that
PTH induces release of calcium from intracellular stores. Thapsigargin
treatment by itself induced transient MAP kinase phosphorylation (Fig.
3B), showing that, also in CHO-R15 cells, a strong increase
in intracellular calcium can be sufficient to activate MAP kinase. To
determine whether an increase in calcium levels is necessary for the
activation of MAP kinase by PTH, we prevented both calcium release and
calcium influx by preincubating the cells with EGTA followed by
thapsigargin. 30 min after the addition of these reagents, the
phosphorylation of MAP kinase had returned to basal levels. Subsequent
addition of PTH(1-34) stimulated MAP kinase phosphorylation to a
similar level as in control cells, indicating that an increase in
intracellular calcium levels is not required for MAP kinase activation
by PTH.
Another GPCR-mediated event, described as being involved in the
activation of MAP kinase, is the release of G subunits (8). To
examine a possible role of G
subunits in the PTH-induced MAP
kinase activation in CHO-R15 cells, we expressed G
t to
sequester G
subunits after they are released from G proteins on
receptor stimulation (8). Fig. 4 shows that
G
t expression efficiently inhibits the activation of a
cotransfected, hemagglutinin-tagged MAP kinase (HA-MAP kinase) (27) by
LPA, showing that G
subunits are indeed efficiently sequestered
(9, 13). However, PTH(1-34)-induced MAP kinase activation is not
affected, suggesting that this is not dependent on the release of
G
subunits.
Elevation of Intracellular cAMP Levels Induces MAP Kinase Activation in CHO Cells and Is Involved in the PTH-induced MAP Kinase Activation
PTH is a strong activator of adenylate cyclase in
CHO-R15 cells (data not shown). Elevation of intracellular cAMP levels
inhibits MAP kinase activation in many cell types (17), and we have
previously shown that it is involved in the inhibition of MAP kinase by
PTH in osteosarcoma cells (24). We were therefore surprised to find that elevation of intracellular cAMP levels by addition of forskolin or
the cell-permeable cAMP analogue 8-bromo-cAMP induced a transient phosphorylation of MAP kinase in CHO-R15 cells (Fig.
5A). The kinetics and the quantity of this
phosphorylation were comparable with the phosphorylation induced by
PTH(1-34) (Fig. 1A). Also, in wild-type CHO cells (CHO-K1),
elevation of cAMP levels resulted in MAP kinase phosphorylation (Fig.
5B). To examine whether Gs-induced cAMP
formation is sufficient for MAP kinase activation, we measured the
activation of MAP kinase by a typically Gs-coupled
receptor, the -adrenergic receptor. Addition of isoproterenol to CHO
cells stably transfected with the
2-adrenergic receptor
(CHO-
2) (37) resulted in a phosphorylation of MAP kinase with the
same quantity and kinetics as the phosphorylation induced by PTH in
CHO-R15 cells (Fig. 5C). 8-Bromo-cAMP also induced
phosphorylation of MAP kinase in these cells (not shown). Thus,
elevation of cAMP levels through activation of Gs is
sufficient to explain MAP kinase activation by PTH in CHO-R15 cells. To
determine whether elevation of cAMP could account for MAP kinase
activation by PTH, we measured activation of MAP kinase after prolonged
incubation (2 h) with forskolin. This resulted in a sustained PKA
activation (Fig. 6B), whereas MAP kinase
activity had returned to basal levels (Fig. 6A). Subsequent
stimulation with insulin or TPA resulted in phosphorylation of MAP
kinase to a comparable level as in nonpretreated cells, whereas
addition of 8-bromo-cAMP or PTH(1-34) no longer induced phosphorylation of MAP kinase (Fig. 6A). 8-Bromo-cAMP or PTH
had no or little effect on PKA activity in cells preincubated with forskolin (Fig. 6B). Similar results were obtained when
cells were incubated overnight with cholera toxin (data not shown). Importantly, preincubation with forskolin had no effect on the PTH(1-34)-induced calcium release (Fig. 6C), showing that
PTH/PTHrP receptor functioning was not impaired. Taken together, these
results suggest that the activation of MAP kinase by PTH is mediated
solely by elevation of intracellular cAMP levels.
cAMP and PTH Activate MAP Kinase via a Ras-independent Pathway
Common pathways for the activation of MAP kinase by both
protein tyrosine kinase receptors and GPCRs involve the activation of
Ras (17). To examine the involvement of Ras in the activation of MAP
kinase by PTH, we interfered with Ras-mediated signaling by
overexpression of a dominant-negative form of Ras,
RasAsn-17 (38). This was performed either by infection of
the cells with recombinant vaccinia virus expressing
RasAsn-17, to interfere with endogenous Ras molecules, or
by cotransfection of RasAsn-17 and HA-MAP kinase.
Stimulations with insulin and TPA were used as controls for,
respectively, Ras-dependent and -independent activation of
MAP kinase (38, 39). Infection with wild-type vaccinia virus had no
effect. As expected, expression of RasAsn-17 completely
inhibited the activation of MAP kinase by insulin, whereas TPA-induced
MAP kinase activation was not affected (Fig. 7A). Interestingly, forskolin and PTH(1-34)
also were still able to induce MAP kinase activation. Similar results
were seen with transfected RASAsn-17 on the activation of
cotransfected HA-MAP kinase (Fig. 7B). These results suggest
the existence of a Ras-independent MAP kinase-activating pathway in CHO
cells, which involves elevated cAMP levels and can be triggered by the
PTH/PTHrP receptor.
PTH Activates MAP Kinase in PYS-2 Cells
To test the relevance
of our findings for PTH/PTHrP receptor signaling, we tested several
cell lines that express the receptor endogenously. Fig.
8 shows that PTH(1-34) can also induce MAP kinase
activation in one such cell line, parietal yolk sac carcinoma (PYS-2)
cells (40). This activation was not affected by prolonged TPA
treatment, suggesting that TPA-sensitive PKC is not involved. PTH is a
strong inducer of cAMP formation in PYS-2 cells (40), and forskolin
induced an activation of MAP kinase in these cells as well, suggesting
a comparable mechanism for PTH-induced MAP kinase activation in PYS-2
and CHO-R15 cells.
In the present study, we show that stimulation of the PTH/PTHrP receptor induces activation of MAP kinase in CHO-R15 and PYS-2 cells. PTH-induced MAP kinase activation was not dependent on PLC-mediated events but appeared to be mediated by elevation of cAMP levels and to occur in a Ras-independent fashion.
Signaling via the PTH/PTHrP receptor involves the activation of at
least two G proteins, Gs and Gq. We have
recently shown that PTH inhibits the activation of MAP kinase in UMR
106 and ROS 17/2.8 cells through activation of PKA (24). Although
PLC--mediated events can induce MAP kinase activation (11, 30, 31,
34), the activation of PLC-
by PTH was apparently not sufficient to affect MAP kinase activity in these cells. It has been reported that
the efficiency of coupling of the PTH/PTHrP receptor to Gq and PLC-
is related to receptor density (29). Because the PTH/PTHrP receptor couples strongly to PLC-
in CHO-R15 cells, most likely because of the high levels of receptor expression, we examined whether
this might explain the differences between the action of PTH on MAP
kinase in CHO-R15 and osteoblast-like cells.
Our data suggest that typical PLC--mediated events, such as release
of calcium from intracellular stores and activation of PKC, are not
involved in the activation of MAP kinase by PTH. Thapsigargin treatment
prevented the PTH-induced calcium response but had no effect on the
activation of MAP kinase by PTH, suggesting that the calcium increase
is not necessary for the PTH-induced MAP kinase activation.
Nevertheless, the small increase in calcium observed with PTH in
untreated cells could still be sufficient for MAP kinase activation.
However, since a combined treatment with PTX and TPA completely blocked
the thrombin-induced MAP kinase activation (Fig. 2), whereas the
thrombin-induced calcium increase was not affected (not shown), and
since the calcium response to PTH is weaker then the one observed with
thrombin, this suggests that the calcium response to PTH is not
sufficient to activate MAP kinase and that a strong response, like that
with thapsigargin, is needed to activate MAP kinase. This is supported
by the observation that prolonged cAMP elevation completely abolished
MAP kinase activation by PTH without affecting the calcium response. An
essential role of PKC was excluded by the observation that
down-modulation of phorbol ester-sensitive PKC did not affect the
activation of MAP kinase by PTH. These data suggest that the
PTH-induced MAP kinase activation is not depending on PLC-
activity.
Other described intermediates between MAP kinase activation and GPCRs
are G subunits. The action of Gi on MAP kinase is established to be fully dependent on G
subunits (8-11, 13). Studies concerning the role of G
subunits in MAP kinase
activation via Gs have produced contradictory results. It
was demonstrated that activation of MAP kinase by the
Gs-coupled
-adrenergic receptor in COS-7 cells is
mediated by G
subunits and is fully dependent on Ras (14).
However, others reported that expression of the Gs-coupled
D1A dopamine receptor in COS-7 cells did not lead to activation of Ras (9). Studies on the activation of MAP kinase by
Gq-coupled receptors have produced contradictory results as well. It was reported that activation of MAP kinase by the M1 acetylcholine receptor occurred in a G
- and
Ras-dependent manner (10), while others showed that
triggering of the M1 acetylcholine receptor, the
1B-adrenergic receptor, or the bombesin receptor resulted in MAP kinase activation that was independent of G
subunits (8, 9, 11) and mediated by PKC in a Raf-dependent but Ras-independent manner (11). In this study, we show that sequestering of G
by overexpressed G
t blocked
LPA-induced MAP kinase activation but did not inhibit the action of
PTH, suggesting that the activation of MAP kinase by PTH is not
dependent on the release of G
subunits. As for most pathways
involved in receptor-mediated MAP kinase activation, the G
subunit-mediated activation of MAP kinase also depends on Ras (9-12,
13, 14). Here we show that inhibition of Ras-mediated signaling by
overexpression of RASAsn-17 completely blocks
insulin-induced MAP kinase activation, whereas it does not interfere
with PTH-induced MAP kinase activation. This suggests that Ras is not
involved in the activation of MAP kinase by PTH. Since expression of
RasAsn-17 inhibits the activation of Ras and not the basal
levels of Ras activity (43), it is still possible that PTH acts in
cooperation with basal Ras activity, as has been suggested for the
activation of MAP kinase by PKC (17).
The time course and extent of MAP kinase activation by cAMP and PTH
were identical, and, importantly, both were shown to be independent of
Ras. Sustained elevation of cAMP levels by forskolin or cholera toxin
prevented activation of MAP kinase by cAMP or PTH, whereas insulin- or
TPA-induced MAP kinase activation was not affected. This suggests that
elevation of cAMP levels is the sole mediator of PTH on MAP kinase. The
PTH-induced calcium transient was, under these conditions, similar to
that in control cells. This shows that PTH/PTHrP receptor activation
was not impaired, at least with respect to PLC- activation. Taken
together, these results strongly suggest that PTH-induced MAP kinase
activation is mediated by elevation of intracellular cAMP levels.
Numerous reports have documented effects of cAMP on MAP kinase activity. The mechanism involved heavily depends on the cell type studied. It is well established that elevation of intracellular cAMP levels inhibits MAP kinase activation in fibroblasts, arterial smooth muscle cells, adipocytes, and osteoblasts (17, 24). It was demonstrated that PKA interfered with Ras-mediated activation of MAP kinase at the level of Raf-1 (44). Activation of MAP kinase by cAMP was first reported in COS and PC12 cells (8, 16). A number of recent reports demonstrated the same phenomenon in other cell types, e.g. Swiss-3T3, melanoma, pituitary, and ovarian granulosa cells (45-48). The mechanism of cAMP-mediated MAP kinase activation, however, is not clear. We show here that elevation of cAMP levels leads to activation of MAP kinase in CHO and PYS-2 cells as well. We draw the following conclusions about the mechanism of cAMP-induced MAP kinase activation from the data presented in this study. (i) Activation of MAP kinase by cAMP is not inhibited by expression of RasAsn-17, indicating that Ras is not involved. This argues either for a site of action downstream of Ras in the Ras-Raf-MEK-MAP kinase cascade or a parallel pathway. (ii) A sustained elevation of cAMP levels leads to down-modulation of cAMP-induced MAP kinase activity. However, insulin and TPA are still fully capable of activating MAP kinase, showing that an effector in the MAP kinase-activating pathway downstream of cAMP is inhibited or down-modulated, which is not essential for the activation of MAP kinase by insulin or TPA. The observation that PKA is still maximally active suggests that this effector is not PKA. (iii) In fibroblasts, a short or prolonged elevation of cAMP levels inhibits MAP kinase activation at the level of Raf-1 (17, 49). In cell types in which cAMP stimulates MAP kinase activity, such as PC12 cells, it has been shown that Raf-1 is inhibited by cAMP elevation (18, 45, 50). Nevertheless, other factors under these circumstances are still capable of activating MAP kinase, suggesting that there is a Raf-1-independent pathway leading to MAP kinase activation. Here we show that, also in CHO cells, even after prolonged elevation of cAMP, the activation of MAP kinase by TPA and insulin was not inhibited, suggesting the existence of a Raf-1-independent pathway as well. Whether MEK and MEK kinases, such as Raf-1 and B-Raf, are involved in the activation of MAP kinase by cAMP in CHO cells remains to be determined.
Although the inhibitory action of cAMP on MAP kinase activation has been shown to correlate with the cAMP-induced inhibition of cell growth, the implication of cAMP-induced MAP kinase activation is less clear. The activation of MAP kinase by cAMP has been suggested to be involved in the neuronal differentiation of PC12 cells (3, 16, 51, 52) and the melanogenesis in melanoma cells (46). In light of these observations it is interesting that PYS-2 cells resemble parietal endoderm cells (40). Studies with embryonic stem cells and F9 embryonal carcinoma cells have established that addition of retinoic acid together with PTH or dibutyryl cAMP induces differentiation to a parietal endoderm-like phenotype (40, 53, 54). It will be interesting to determine whether activation of MAP kinase by cAMP is involved in the PTH-induced differentiation to parietal endoderm.
In conclusion, our data illustrate that triggering of the PTH/PTHrP receptor activates MAP kinase in CHO-R15 and PYS-2 cells. The PTH-induced MAP kinase activation did not depend on PLC-mediated events but appeared to be mediated by elevation of cAMP levels. Although the majority of receptor-mediated activations of MAP kinase occurs in a Ras-dependent fashion, we showed that Ras is not involved in the activation of MAP kinase by cAMP and PTH. This suggests the existence of a Ras-independent pathway involving elevated cAMP levels that is triggered by the PTH/PTHrP receptor. Since we have previously shown that the PTH-induced inhibition of MAP kinase activation in osteosarcoma cells involves elevation of cAMP levels as well, PTH/PTHrP receptor signaling to MAP kinase is correlated with the effect of cAMP on MAP kinase in a cell type-specific fashion. This might determine the effect of PTH on MAP kinase-dependent cellular responses, such as proliferation and differentiation.
We thank Drs. J. L. Bos and B. M. T.
Burgering for the RasAsn-17-expressing vaccinia virus and
generous supply of antibodies, A-B. Abou-Samra (Massachusetts General
Hospital and Harvard Medical School, Boston, MA) for the CHO-R15 cells,
P-O. Couraud (Université Paris VII, Paris, France) for the
CHO-2 cells, H. R. Bourne (University of California, San Francisco,
CA) for the G
t expression construct, and J. Pouysségur (Université de Nice, Nice, France) for providing the p44 HA-MAP kinase expression construct. We thank J. den Hertog for
critically reading the manuscript.