(Received for publication, April 11, 1995; and in revised form, June 30, 1995)
From the
The rat pheochromocytoma (PC12) cell line is a model for studying the mechanism of growth factor action. Both epidermal growth factor and nerve growth factor stimulate mitogen-activated protein (MAP) kinase in these cells. Recent data suggest that the transient activation of MAP kinase may trigger proliferation, whereas sustained activation triggers differentiation in these cells. We have tested this model by asking whether agents that stimulate MAP kinase without inducing differentiation can act additively to trigger differentiation. Neither forskolin nor epidermal growth factor can stimulate differentiation, yet both activate MAP kinase in these cells. Together, their actions on MAP kinase are synergistic. Cells treated with both agents differentiate, measured morphologically and by the induction of neural-specific genes. We propose that cellular responses to growth factor action are dependent not only on the activation of growth factor receptors by specific growth factors but on synchronous signals that may elevate MAP kinase levels within the same cells.
In many cells, both proliferation and differentiation are
triggered by specific growth factors(1) . Despite differences
in their physiological actions, many growth factors engage similar
intracellular signaling pathways, initiated by the autophosphorylation
of specific transmembrane receptors. This phosphorylation recruits
multiple signaling molecules into a membrane-associated complex that
includes the GTPase p21. Activation of Ras
initiates a cascade of phosphorylation and activation of protein
kinases, including the activation of MAP kinase(2) . Growth
factor activation of MAP kinase is required for proliferation in many
cell types(3) .
PC12 cells have provided researchers with
the best studied example of dual regulation within a single cell
through the distinctive actions of NGF ()and
EGF(5) . Both signals are mediated through a family of
receptors with intrinsic tyrosine kinase activity yet display
dramatically different effects on neural development. NGF-induced
differentiation of PC12 cells is characterized by the prolongation of
neurites, the induction of neural markers, and the cessation of cell
division. In contrast, EGF stimulates PC12 cell proliferation.
Interestingly, both EGF and NGF activate a receptor tyrosine kinase to
phosphorylate and activate similar intracellular substrates including ras and MAP kinase. However, these treatments stimulate
distinct physiological responses, resulting in proliferation and
differentiation, respectively(5) . This differentiation is
characterized by the elongation of neuritic processes and the induction
of specific genes, including transin, that are involved in maintaining
neuronal phenotypes(6, 7) . Though ras activation is necessary and sufficient for this
differentiation(8, 9) , the requirement of MAP and ERK
kinase activation has only recently been established(10) .
Despite the contrasting actions of EGF and NGF, the points of divergence accounting for the expression of such different cellular phenotypes have not been determined. Recent data suggest that the duration of MAP kinase activation may dictate proliferative and differentiative responses in PC12 cells(11) . In addition, it has been shown that sustained activation of MAP kinase (extracellular signal-regulated kinase (ERK)) by NGF in these cells allows for the nuclear translocation of ERKs(12) . This may initiate a program of differentiation and growth arrest, presumably through the action of nuclear substrates of MAP kinase or associated kinases.
We have tested this model by asking whether agents that stimulate MAP kinase without inducing differentiation can act additively to trigger differentiation. cAMP has been shown to activate MAP kinase in PC12 cells without differentiating these cells(13, 14, 15) . Here we show that stimulation of cAMP levels by forskolin induces a transient stimulation of MAP kinase activity and a persistent localization of MAP kinase (ERK1) within the cytoplasm. The application of forskolin and EGF to these cells altered the physiological action of both agents by inducing differentiation as judged by changes in morphology and gene expression. This differentiation was associated with a sustained activation and nuclear localization of MAP kinase. The morphological actions of EGF and forskolin were blocked by specific inhibition of MAP kinase activation by MAP kinase phosphatase-1, demonstrating that the synergistic effects of EGF and cAMP are mediated by the activation of MAP kinase. We propose that cellular responses to growth factor action are dependent not only on the activation of growth factor receptors by specific growth factors but on synchronous signals that elevate intracellular signals like cAMP that can activate MAP kinase within the same cells. Because cAMP is regulated by hormonal stimulation, these data suggest that the specificity of growth factor action depends not only on signals generated by growth factor receptors but also on the hormonal milieu in which growth factors act.
Examination of the magnitude and time course of MAP kinase activation in PC12 cells by EGF and NGF suggests how one intracellular pathway can stimulate both differentiation and proliferation in the same cell. Proliferative agents like EGF activate MAP kinase rapidly and transiently. During this brief activation, MAP kinase remains in the cytoplasm in PC12 cells(2, 12) . In contrast, differentiation by NGF is characterized by prolonged activation and nuclear translocation of MAP kinase(11, 12) , where it may regulate differentiation-specific genes through the phosphorylation of nuclear transcription factors(2) .
Forskolin induces an activation of MAP kinase that is lower in magnitude than that induced by NGF (13, 14, 15) and does not stimulate differentiation(13) . Still, cAMP is synergistic with NGF (and fibroblast growth factor) to potentiate differentiation, measured morphologically (16) and by the induction of specific genes(17, 18) . Therefore, it is possible that MAP kinase activation must reach a threshold of sufficient magnitude and duration to trigger differentiation in these cells.
The possibility that a threshold of MAP kinase activation exists, above which a cell is committed to a pathway of differentiation, could have profound implications for the physiological regulation of growth and differentiation in normal cells. Signals that activate MAP kinase to levels that are not sufficient to trigger differentiation when acting alone might be sufficient to trigger differentiation when acting together. To test this hypothesis, we examined the physiological response of PC12 cells to EGF and agents that stimulate cAMP.
Forskolin, an agent that stimulates adenylyl cyclase in these cells(21) , did not stimulate neurite formation (Fig. 1), as previously reported(13, 22) . Simultaneous treatment with EGF and forskolin produced morphological changes including an outgrowth of neurites indistinguishable from those induced by NGF alone (Fig. 1). The cAMP analog 8-aminohexylamino-cAMP (23) but not 3-isobutyl-1-methylxanthine gave similar results (data not shown). Neurites were first observed in greater than 50% of cells following 24 h of treatment, and they increased in density and length by 48 h. These changes but not those induced by NGF could be blocked by the protein kinase A inhibitor H89(24) , confirming that NGF's actions do not require protein kinase A, as previously proposed ( (25) and Fig. 1). Similar results were seen with another PC12 cell line, P19 (kindly provided by Louis Reichardt; data not shown).
Figure 1: Photomicrograph of PC12 cells demonstrating that EGF/forskolin induces morphological changes in PC12 cells. PC12 cells were grown to 10% confluency on collagen-coated slides and then left untreated (A) or incubated with forskolin (B), EGF (C), NGF (D), NGF plus H89 (D, inset), EGF/forskolin (E), or EGF/forskolin plus H89 (E, inset). Phase contrast micrographs were taken with a Leitz Dialux 22EB microscope. The bar represents 20 µm.
The requirement for MAP kinase activation was investigated by transfecting three cDNAs whose expression inhibits MAP kinase activity. One cDNA encodes the MAP kinase phosphatase-1. MAP kinase phosphatase-1 is a dual specificity phosphatase that inactivates MAP kinase by specifically dephosphorylating both MAP kinase isoforms ERK1 and ERK2(26) . Transient expression of MAP kinase phosphatase-1 selectively inactivates MAP kinase in many cell types(27, 28, 29) . We have shown that in PC12 cells MAP kinase phosphatase-1 inactivates MAP kinase and blocks MAP kinase-dependent transcription(30) . The other two cDNAs used in these studies encode interfering mutants of ERK1 and ERK2 that block endogenous MAP kinase activity by acting as dominant negative mutants (referred to here as dn ERKs) (32) (kindly provided by Melanie Cobb).
To examine the inhibition of neurite outgrowth by MAP
kinase phosphatase-1, cells were transfected with either a plasmid
pcDNA3 (Invitrogen) containing the full-length mouse MAP kinase
phosphatase-1 (kindly provided by Nicholas Tonks) (pMKP-1) under the
control of a cytomegalovirus immediate early promoter or vector alone
(pcDNA3). Cells were also transfected with a plasmid directing the
expression of the lacZ gene product -galactosidase under
the control of an RSV promoter (RSV-
-galactosidase) (32) as a marker for transfected cells. After 24 h, cells were
serum-starved for an additional 24 h and treated with NGF or
EGF/forskolin as described (19) . After incubation for an
additional 48 h, the cells were stained for
-galactosidase using
histochemical methods(31) . Using this technique, the
expression of
-galactosidase identified co-transfected cells (data
not shown). The expression of MAP kinase phosphatase-1 blocked the
morphological changes induced by NGF and EGF/forskolin (Fig. 2, A and B, and 3). This inhibition reduced the fraction
of neurite-containing blue cells to less than 20% without affecting the
differentiation of cells that did not express
-galactosidase (Fig. 3). This demonstrates that EGF and agents that stimulate
cAMP levels require MAP kinase to induce morphological changes. Similar
results were seen in cells co-transfected with plasmids expressing
interfering mutants of ERK1 and ERK2 (dn ERKs) (Fig. 3).
Figure 2:
Photomicrograph of transfected PC12 cells
stained for -galactosidase activity. PC12 cells were transfected
with 5 µg of RSV-
-galactosidase and either 30 µg of pMKP-1 (A and B) or 30 µg of PEXV3 MAPKK1 (C)
and either treated with NGF (A) or EGF/forskolin (B)
or left untreated (C). Note that treatments with either NGF or
EGF/forskolin induced neurites in non-transfected cells surrounding the
blue cells in A and B, and in contrast, the
non-transfected cells surrounding the blue cells in C have not
differentiated. This experiment was performed three times with similar
results as shown in Fig. 3.
Figure 3:
The percentage of neurites in PC12 cells
transfected with cDNAs that modulate MAP kinase activation. Cells were
transfected with 5 µg of RSV--galactosidase and 30 µg of
pMKP-1, 15 µg of both dn ERK1 and ERK2, or 30 µg of vector
pcDNA3 with and without 30 µg of pEXV3 MAPKK1. Subsequently, cells
were treated with NGF or EGF/forskolin or left untreated as indicated.
The percentage of cells with neurites is shown. White bars represent RSV-
-galactosidase-negative cells, and shaded
bars represent RSV-
-galactosidase-positive cells. Note that
the extension of neurites in untransfected cells (white bars)
is unaffected by the conditions of the transfection in all assays. Each bar represents a total of at least 100
cells.
As a
control for the previous experiment, cells were transfected with pEXV3
MAPKK1 (kindly provided by Christopher Marshall). pEXV3 MAPKK1 encodes
a constitutively active mutant of MAP and ERK kinases that
differentiates PC12 cells when introduced via microinjection (10) . Likewise, transient transfection of this plasmid also
stimulated neural differentiation (Fig. 2C). Cells were
co-transfected with 5 µg of RSV--galactosidase and pEXV3
MAPKK1, and the expression of
-galactosidase was detected by the
presence of blue cells, as described above. Greater than 80% of blue
cells displayed neuritic processes (Fig. 2C and 3). In
contrast, less than 20% of non-blue cells displayed morphological
changes, demonstrating that cells expressing RSV-
-galactosidase
also expressed the co-transfected plasmid. The differentiation seen
following transfection with pEXV3 MAPKK1 was blocked in
co-transfections with cDNAs encoding dn ERK1 and dn ERK2 or MAP kinase
phosphatase-1 (Fig. 2A and Fig. 3).
Expression of RSV--galactosidase alone did not stimulate
neurites, nor did it interfere with the extension of neurites (Fig. 3). These results complement those of Cowley et
al.(10) , who showed that microinjection of PEXV3 MAPKK1
is sufficient for neuronal differentiation in PC12 cells. They also
reported that injection of inactivating mutants of MAP and ERK kinases
could block NGF-induced morphological changes in PC12
cells(10) . These studies and those presented here strongly
suggest that the activation of MAP kinase, as well as MAP and ERK
kinases, is required for neuronal differentiation.
Neuronal differentiation of PC12 cells by NGF is associated with the induction of both immediate early genes including egr-1(33) and fos(34) , and late genes including scg10(35) , the neural adhesion marker L1(36) , and the metalloprotease transin (stromelysin)(6) . Transin encodes a metalloprotease that is expressed in neuronal and non-neuronal cells. Its expression in the developing nervous system has suggested a role in growth cone guidance and axonal path finding (37) and correlates with neuronal differentiation in PC12 cells(20, 38) . To examine whether treatment with EGF/forskolin was able to induce transin expression, RNA was isolated from treated and untreated PC12 cells and subjected to Northern blotting using an antisense RNA probe corresponding to the transin cDNA(20) . As shown in Fig. 4A, EGF/forskolin, as well as NGF, induced the expression of a 1.9-kilobase band corresponding to the rat transin mRNA(20) . In contrast, neither forskolin nor EGF alone induced transin expression, as described previously(20) . Surprisingly the level of transin expression appeared to be significantly higher with EGF/forskolin (28-fold over basal) than with NGF (8-fold over basal) (Fig. 4A).
Figure 4: NGF and EGF/forskolin stimulate transcription from a transin promoter. A, NGF and EGF/forskolin stimulate transin mRNA levels. An autoradiograph of a Northern blot detecting transin mRNA demonstrating transin mRNA induction by EGF plus forskolin in PC12 cells is shown. Lane 1, untreated cells (U); lane 2, EGF (E); lane 3, forskolin (F); lane 4, EGF/forskolin (E/F); lane 5, NGF (N). The expected size of the transin band is indicated (1.9 kb). B, induction of chloramphenicol acetyltransferase activity expressed from a transin promoter. Cells were transfected with 5 µg of the plasmid pTRCAT and 30 µg of vector pcDNA3 alone, 5 µg of pEXV3 MAPKK1, 30 µg of pMKP-1, or 30 µg of pCaN420 and stimulated with NGF or EGF/forskolin or left untreated as indicated. Basal represents the chloramphenicol acetyltransferase activity seen in unstimulated cells transfected with pTRCAT and vector. Each value represents the average with standard error of at least three experiments and is presented as fold increase over basal levels.
The induction of transin expression by NGF is dependent on upstream activators of MAP kinase including ras(6) and may be a marker for neuronal differentiation through the MAP kinase cascade. To demonstrate a requirement of MAP kinase for the induction of transin, we transfected a reporter gene encoding chloramphenicol acetyltransferase under the control of 750 base pairs of the transin promoter (pTRCAT, kindly provided by Gary Ciment). The expression of pTRCAT in PC12 cells qualitatively reflects NGF's effects on transin expression through cis-acting promoter elements contained on the plasmid(19) . Transfections and chloramphenicol acetyltransferase assays were performed as described(19) . As shown in Fig. 4B, NGF treatment for 24 h induced a 5-fold stimulation of chloramphenicol acetyltransferase activity, and transfection of pEXV3 MAPKK1 induced a 16-fold stimulation of chloramphenicol acetyltransferase activity. Both activities were blocked by co-transfection of MAP kinase phosphatase-1. Treatment with EGF/forskolin stimulated a 6-fold increase in chloramphenicol acetyltransferase activity that was also blocked by co-transfection with pCMV-MKP-1 but not by pCaN420, encoding a constitutive mutant of calcineurin(39) , a serine/threonine phosphatase with no known activity against MAP kinase (provided by Thomas Soderling, Vollum Institute). These data suggest that MAP kinase activation participates in transin induction by these agents.
To examine the kinetics of MAP kinase activation by EGF/forskolin, PC12 cells were treated with both agents and lysed at the indicated times. Cells lysates were subjected to immunoprecipitation with ERK1 antisera, and MAP kinase activity was measured within the immune complexes. EGF stimulation produced a rapid, transient activation that reached a maximum within 5 min and returned to low levels after 30 min. Forskolin treatment produced a slow rise in MAP kinase activity, reaching a maximum at 20 min. At all time points of forskolin treatment, MAP kinase activity was lower than the corresponding level induced by NGF and, like EGF, returned to low levels for the subsequent time points examined (Fig. 5). In contrast, treatment of PC12 cells with EGF/forskolin produced a substantial and sustained activation of ERK1 that paralleled that seen with NGF (Fig. 5). Nuclear localization of ERK1 was correlated with its sustained activation (Fig. 6). Only those cells treated with either NGF or EGF/forskolin (but not cells treated with EGF or forskolin alone) showed detectable nuclear staining.
Figure 5:
Time
course of MAP kinase activation by cAMP, EGF, NGF, and EGF/forskolin.
Cells were treated with agents for the indicated times and ERK-1
activity was assayed and measured as described. Basal activity
represents the activity in unstimulated cells. Activity is expressed as
fold increase over basal (time 0) to allow direct comparison between
the following treatments: forskolin (), EGF (
), NGF
(
), and EGF/forskolin (
).
Figure 6: Photomicrograph of 10-µm sections of paraffin-embedded cell blocks demonstrating immunohistochemical detection of ERK1(39) . PC12 cells were treated with various agents for 90 min. 1, NGF; 2, EGF; 3, forskolin; 4, EGF/forskolin. Nuclei are counterstained with hematoxylin (purple), and cytoplasmic regions are counterstained with eosin (pink). The reddish orange histological reaction product represents localization of ERK1.
Using morphological, molecular, and biochemical criteria, we have established that forskolin can convert the physiological response of EGF from one of proliferation to one of differentiation. Using these same criteria, neither EGF nor forskolin alone produced a differentiated response. In all assays, co-stimulation with EGF and forskolin produced responses similar to those of NGF. In addition we have shown that both treatments (EGF/forskolin and NGF) require MAP kinase. It has been proposed that differentiation of PC12 cells by growth factors requires a threshold of MAP kinase activity(11, 12) . The notion of a threshold for differentiation is supported by studies of PC12 cells that have been genetically altered to express high levels of the EGF receptor (40) or the adapter protein Crk that couples this receptor to ras activation(41) . Both alterations result in cell lines displaying enhanced MAP kinase activation and neuronal differentiation in response to EGF. In addition, in contrast to forskolin, the long acting non-hydrolyzable cAMP analog 8-(4-chlorophenylthio)-cAMP can stimulate sustained activation of MAP kinase to levels similar in magnitude and duration to that of NGF and can induce PC12 differentiation(42) . The studies presented here are consistent with this notion that the level of MAP kinase activity may dictate the physiological responses to MAP kinase activation. In addition, these studies demonstrate that nuclear translocation of MAP kinase is associated with prolonged activation of MAP kinase, suggesting that the translocation of MAP kinase or associated kinases (2) may activate a program of differentiation and/or growth arrest.
The mechanism by which cAMP stimulates MAP kinase in PC12 cells is not completely understood, although this stimulation has been reported to involve the activation of MAP and ERK kinases(13) . Although we have shown that cAMP-mediated activation of MAP kinase can augment EGF signaling, cAMP's activation of other pathways may be important as well. For example, forskolin significantly increases the stimulation of transin mRNA levels by both NGF (data not shown) and EGF (Fig. 4A) to a degree that far exceeds the cooperativity of forskolin with these factors in the stimulation of MAP kinase (Fig. 5). Protein kinase A is not required for NGF's induction of neural differentiation (Fig. 1D, inset). Similar data have been used to suggest that cAMP-dependent gene transcription is not important in mediating NGF's actions(25) . cAMP-dependent gene transcription involves the phosphorylation of the transcription factor CREB (cyclic AMP responsive element binding protein). However, it has recently been shown that CREB can be activated by NGF in PC12 cells through a protein kinase A-independent pathway downstream of ras(43) and possibly downstream of MAP kinase as well. Therefore, although sustained activation of MAP kinase may be sufficient for differentiation of PC12 cells(44) , the involvement of CREB-dependent gene transcription cannot be ruled out.
The studies presented here demonstrate that co-stimulation by
physiological agents can produce cooperative effects on MAP kinase that
are sufficient to induce novel biological responses. The ability of
cAMP to augment EGF's activation of MAP kinase to trigger
differentiation has profound implications in the hormonal regulation of
growth and differentiation. Proliferation and differentiation appear to
be mediated, in part, by a common pathway. The physiological response
of this pathway may be dramatically influenced by additional hormonal
signals. For example, EGF receptors are expressed in cells throughout
the developing brain, where they are thought to exert proliferative
effects during development(45) . These cells also express
multiple receptors for hormones that are coupled to adenylyl cyclase
activation, therefore their physiological response to EGF may be
dictated by the synchronous contributions of hormonal signals. cAMP may
regulate growth factor action in non-neuronal cells as well. For
example, cAMP can stimulate activation of MAP kinase in cardiac
myocytes (46) and S49 lymphocytes. ()This action of
cAMP may synergize with growth factors to mediate hypertrophic
responses in the heart (47) and/or induce tolerance in
stimulated T cells(48) .