©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cyclic Adenosine Monophosphate Can Convert Epidermal Growth Factor into a Differentiating Factor in Neuronal Cells (*)

(Received for publication, April 11, 1995; and in revised form, June 30, 1995)

Hong Yao (1) (4) Kirstin Labudda (2) Caroline Rim (4) Paola Capodieci (5) Massimo Loda (5) Philip J. S. Stork (4) (3)

From the  (1)Departments of Microbiology and Immunology, (2)Biochemistry, and (3)Pathology and (4)The Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201 and the (5)Department of Pathology, Deaconess Hospital, Boston, Massachusetts 02138

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 (^1)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.


MATERIALS AND METHODS

Cell Culture

PC12-GR5 cells (provided by Rae Nishi, Oregon Health Sciences University) were maintained at 37 °C in a 5% CO(2) humidified atmosphere in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated horse serum and 5% fetal calf serum (Life Technologies, Inc.). 16 h prior to treatments, cells were switched to serum-free medium. 50 ng/ml NGF (Boehringer Mannheim), 100 ng/ml EGF (Boehringer Mannheim), 10 µM H89 (Calbiochem), and 10 µM forskolin (Calbiochem) were added as indicated, and the cells were incubated for an additional 48 h. The extension of neurites was assayed as described previously(4) .

Plasmids and Transient Transfection Assays

For all transfections, the calcium phosphate precipitation method was used (Life Technologies, Inc.). One day prior to transfection, cells were plated at a density of 2 10^6 cells/plate as described (19) . The following day they were transfected with combinations of the following plasmids: 5 µg of RSV-beta-galactosidase, 30 µg of pMKP-1, 5 µg of pEXV3 MAPKK1, 15 µg of dn ERK1, 15 µg of dn ERK2, 30 µg of pCaN420, and 5 µg of pTRCAT. 4 h following the transfection, the cells were washed in hypotonic buffer (25% glycerol) to facilitate DNA uptake(19) . The cells were maintained in complete media for an additional 24 h and fed with supplemented serum-free media(N2) containing 5 µg/ml insulin, 100 µg/ml transferrin, 30 µM sodium selenite, 100 µM putrescine, and 20 µM progesterone(19) .

MAP Kinase Assays

Treated and untreated PC12 cells (1 10^7 cells) were lysed in 1% Nonidet P-40 lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl(2), 10% glycerol, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 2 mM vanadate). The lysates were centrifuged at 10,000 g for 10 min at 4 °C, and the supernatants were immunoprecipitated with agarose-conjugated anti-ERK1 antibody (anti-ERK1) for 2 h at 4 °C. The immunoprecipitates were assayed for kinase activity by incubating with 25 µg of myelin basic protein and 50 µM 10 µCi of [-P]ATP in 50 µl of buffer containing 40 mM Hepes, pH 7.4, 40 mM MgCl(2), and 0.2 µM ATP for 30 min at 30 °C. The reactions were terminated by adding an equal volume of 2 Laemmli buffer and resolved in a 10% SDS-polyacrylamide gel and then analyzed by autoradiography. The bands were quantitated using a PhosphorImager.

RNA Analysis

Total RNA from PC12 cells treated with NGF, EGF, forskolin, and EGF/forskolin was prepared using guanidium salts, separated by electrophoresis, and transferred to nylon filters. The filters were hybridized at 65 °C with one million cpm of antisense cRNA (1 µg) generated from a riboprobe vector (Bluescript) containing the full-length transin (stromelysin) cDNA (kindly provided by Gary Ciment)(20) . The filters were analyzed by autoradiography.

Immunohistochemical Localization of ERK1

10-cm plates of PC12 cells grown to 50% confluency were treated with NGF, EGF, forskolin, and EGF/forskolin for 90 min. The cells were washed, fixed in 4% formaldehyde (at 4 °C for 5 min), and scraped into 1 ml of phosphate-buffered saline. The suspension of whole cells was centrifuged at 2000 g for 10 min at 4 °C, and the pellet dehydrated in graded ethanols. The cell block was perfused in paraffin and embedded for microtomy. 5-µm sections were mounted on glass slides, pre-treated with trypsin (10 min), and used for immunohistochemical staining with antisera against ERK-1 (Santa Cruz Technology, Santa Cruz, CA; 2 µg/ml, representing a dilution of 1:50). The reaction was carried out in an automated immunohistochemistry instrument, the Ventana 320/ES (Ventana Medical Systems, Tuscon AZ). Antigen-antibody reactions were revealed with standardized development times utilizing the avidin biotin complex method using diaminobenzidine as substrate.


RESULTS

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 beta-galactosidase under the control of an RSV promoter (RSV-beta-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 beta-galactosidase using histochemical methods(31) . Using this technique, the expression of beta-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 beta-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 beta-galactosidase activity. PC12 cells were transfected with 5 µg of RSV-beta-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-beta-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-beta-galactosidase-negative cells, and shaded bars represent RSV-beta-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-beta-galactosidase and pEXV3 MAPKK1, and the expression of beta-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-beta-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-beta-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 (diamond, filled), NGF (black square), 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.




DISCUSSION

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. (^2)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) .


FOOTNOTES

*
This work is supported by National Institutes of Health Grant R01-DK45921-03 and a fellowship from the N. L. Tartar Research Fund (to H. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: NGF, nerve growth factor; EGF, epidermal growth factor; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; dn, dominant negative; RSV, Rous sarcoma virus.

(^2)
P. J. S. Stork, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Gary Ciment and Sunita de Sousa for providing transin probes and technical assistance, Christopher Marshall for providing DNA-encoding constitutively active MAP and ERK kinases, Nicholas Tonks for providing cDNA-encoding MAP kinase phosphatase-1, Anita Misra-Press and Andrey Shaw for helpful discussions, Gary Ciment and Michael Forte for critical reading of the manuscript, and Sheri Medford for secretarial assistance.


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