©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Staurosporine Causes Epidermal Growth Factor to Induce Differentiation in PC12 Cells via Receptor Up-regulation (*)

(Received for publication, November 21, 1994; and in revised form, December 27, 1994)

Simona Raffioni Ralph A. Bradshaw (§)

From the Department of Biological Chemistry, College of Medicine, University of California, Irvine, California 92717

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Although they all utilize tyrosine kinase receptors and activate signaling pathways characterized by a similar set of phosphoproteins, epidermal growth factor (EGF) promotes only cell division while fibroblast growth factor (FGF) and nerve growth factor (NGF) can induce division followed by differentiation in PC12 cells. EGF, in contrast to NGF and FGF, cannot maintain the sustained phosphorylation and activation of mitogen-activated protein (MAP) kinase kinase and MAP kinases, which may account for the difference in phenotypic response. The pretreatment of PC12 cells with staurosporine, a protein kinase inhibitor, causes a substantial increase in both receptor and MAP kinase phosphorylation that results in a differentiative response (neurite proliferation). However, neurites begin to disappear after 3 days, despite the continual presence of EGF, and are largely gone after 5 days, which is not the case with NGF and FGF. Thus, the effect of staurosporine is not permanent. Northern and Western blots indicate that the staurosporine response mainly results from a substantial up-regulation in EGF receptor synthesis, thus providing a much stronger cell surface signal and supporting the view that quantitative rather than qualitative differences distinguish the EGF versus NGF/FGF signaling pathways in these cells.


INTRODUCTION

Polypeptide growth factors interact with plasma membrane-bound receptors to induce signal cascades that begin with protein tyrosine phosphorylation. The activated kinases are either an inherent part of the receptor or associate non-covalently with it as accessory proteins (1, 2) . The pathways that are subsequently stimulated involve a variety of intracellular effector and adaptor proteins and generally result overall in either a mitotic or trophic response(3) . Rat pheochromocytoma-derived PC12 cells (4) have been extensively utilized as a paradigm to deduce the molecular mechanisms that underlie (and distinguish) these two responses, primarily because both division and differentiation can be induced by several well studied factors(5, 6, 7) . Thus, NGF (^1)and FGF (basic or acidic) can promote neurite proliferation while EGF and insulin-like growth factor-I cause only mitosis, despite the fact that all utilize receptors containing a tyrosine kinase effector and activate similar signaling cascades with overlapping sets of phosphorylatable substrates. These observations have raised the question of whether the two responses are induced by fundamentally unique mechanisms or whether quantitative differences, i.e. the level or duration of an essential activated component(s), are responsible(8) .

Although the detailed mechanisms for the induction of differentiation in PC12 cells by either NGF or FGF has not yet been determined, recent findings have underscored the importance of a pathway controlled by p21 that leads to the activation of a kinase cascade involving Raf, MAPK kinase, and MAPK(9, 10, 11, 12, 13, 14, 15, 16) . The adaptor proteins Shc and Grb2 (or Crk) appear to be required to link Ras to the activated receptor (3) via a Ras guanine-nucleotide exchange protein (SOS or C3G). Of particular importance apparently is the sustained activation of MAPK kinase and MAPK and the transfer of the latter to the nucleus (17, 18) . EGF, which can also activate MAPK kinase and MAPK, cannot maintain the necessary level of phosphorylation nor induce nuclear translocation, and it has been suggested that this may result from the rapid internalization of occupied EGFR because of the phosphorylation of Ser-1046/Ser-1047 (by an as yet unidentified kinase)(19) . This would suggest that EGF, which prematurely attenuates its own signal, induces the necessary responses to produce differentiation, but because of the rapid loss of cell surface receptors following ligand binding and the concomitant loss in the transmembrane signal, it cannot do so. In this report, we show that staurosporine, a broad inhibitor of protein kinases, can convert EGF to a differentiative factor for PC12 cells by causing a marked up-regulation of EGFR synthesis as well as by directly affecting receptor phosphorylation/dephosphorylation, thus establishing that the endogenous rat EGFR has the requisite specificity to stimulate both mitotic and differentiative responses in PC12 cells.


EXPERIMENTAL PROCEDURES

Reagents

Mouse EGF was prepared by the method of Savage and Cohen (20) or was obtained from Life Technologies, Inc. Monoclonal anti-rat EGFR antibody (21) was a gift from Maureen O'ConnorMcCourt (National Research Council, Canada). A monoclonal antibody C13 corresponding to residues 996-1022 in the human EGFR (22) and an antibody made against a peptide in the submembrane domain of human EGFR were a gift from Gordon N. Gill (University of California, San Diego). Anti-phosphotyrosine antiserum was obtained from Zymed Labs. Anti-ERK-1 antiserum and Protein-A/G PLUS-agarose were from Santa Cruz Biotechnology, Inc. I-Labeled protein A was purchased from ICN. Staurosporine was from Calbiochem. Molecular weight standards were from Bio-Rad and Life Technologies, Inc. Immobilon P membranes were from Millipore.

Cell Culture

PC12 cells were obtained from E. Shooter (Stanford University) and maintained in culture in 150-cm^2 tissue culture flasks (Costar) in Dulbecco's modified Eagle's medium containing 10% horse serum, 5% fetal calf serum, and 1% Pen-strep solution (Life Technologies, Inc.) (complete medium) in a 5% CO(2) humidified atmosphere.

Neurite Outgrowth Assay

PC12 cells were plated in collagen-coated 6-well plates in complete medium at a density of 0.2 times 10^5 cells/well. After 12-16 h, the medium was changed, and the cells were cultured in the presence of EGF (10 ng/ml) and/or various concentrations of staurosporine. The medium was replaced every other day. Cells were examined for the presence of neurites at various times. Responsive cells were defined as those bearing neurites at least 1 cell diameter in length.

Immunoprecipitation and Immunoblot Analysis

For whole cell lysates, cells were plated in complete medium on either 100- or 150-mm^2 collagen-coated culture dishes and grown to 60-70% confluency. The medium was replaced 16 h before the experiments, and 10 nM staurosporine was added at the same time or for different lengths of time as specified. At the end of this period, cells were treated with EGF (10 ng/ml) for the specified time, then rinsed with cold phosphate-buffered saline containing 1 mM sodium orthovanadate and lysed in cold lysis buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1% Triton X-100, 5 mM EDTA, 200 µM sodium orthovanadate, 50 mM NaF, 30 mM sodium pyrophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) for 15 min on ice(23) . Insoluble material was pelleted in a microcentrifuge at 14,000 rpm for 15 min. Protein concentration was determined with the Bradford colorimetric assay from Bio-Rad. For immunoprecipitations, lysates (2 mg) were incubated with monoclonal anti-rat EGFR antibody for 2 h at 4 °C, and immunoprecipitates were collected using protein A/G-agarose for an additional 2 h. After being washed, the complexes were boiled for 5 min in SDS sample buffer and resolved by 7.5% SDS-PAGE and transferred to Immobilon P membranes by electroblotting at 63 V overnight. The membranes were probed with antibodies as specified and then incubated with I-labeled protein A.

Northern Blot Analysis

A 750-base pair DNA fragment was generated by reverse transcriptase-polymerase chain reaction using total RNA from PC12 cells and oligonucleotides corresponding to amino acids 33-272 of the extracellular domain of rat EGFR(24) . The amplified fragment was subcloned into pGEM-7Zf(+) using the restriction sites BamHI and SphI. The linearized plasmid was used as a template to generate an antisense RNA and was used for hybridization with blots at 65 °C in a hybridization solution containing 5 times SSPE, 50% formamide, 5 times Denhardt, 1% SDS, 10% dextran sulfate, and 100 µg/ml salmon sperm DNA. After 20 h, blots were washed at 67 °C in 0.2 times SSPE containing 0.5% SDS. Subsequently, blots were stripped and reprobed at 42 °C with a 0.74-kilobase BamHI DNA fragment of CHOb (25) used to normalize RNA loading differences.


RESULTS AND DISCUSSION

Staurosporine was initially described as a specific inhibitor of protein kinase C (26, 27) and was subsequently shown to be a highly specific inhibitor of the NGF receptor (as well as other neurotrophin receptors) tyrosine kinase (28, 29, 30) but not other receptor tyrosine kinases, including those for FGF, which can also induce the differentiative response in PC12 cells(31) . Staurosporine has also been shown to have some neurite proliferative effects in PC12 cells at high concentrations(32, 33, 34) . As shown in Fig. 1, at a concentration of 5 times 10M, it does show some differentiative activity in this system. However, the presence of EGF, which alone has no neurite proliferative capabilities, greatly enhances this response. Interestingly, the increase in the EGF response is most pronounced during the first 3 days of treatment and begins to decline thereafter (Fig. 2). The response to staurosporine alone is relatively constant over this time period, suggesting that the principal effect is an augmentation of the EGF signal rather than that induced by staurosporine. The magnitude of the response (particularly during days 1-3) is comparable with that induced by NGF and FGF. However, these factors do not show a diminution of response at later time points(6) , unless the factor is removed.


Figure 1: Dose-dependent induction of neurites in PC12 cells by staurosporine (bullet) and EGF plus staurosporine (). PC12 cells were cultured in the presence of EGF (10 ng/ml) and/or various concentrations of staurosporine. After 24 h of treatment, cells were examined for the presence of neurites. Less than 1% of cells grow neurites in response to EGF. Values are averages of quadruplicate determinations from two to three separate experiments. Verticalbars represent S.E.




Figure 2: The induction of neurite outgrowth by the combined action of EGF and staurosporine in PC12 cells. Cells were cultured in complete medium in the presence of medium alone (CON) or with EGF (10 ng/ml) (EGF), 10 nM staurosporine (ST), or EGF (10 ng/ml) plus 10 nM staurosporine (EGF + ST). The medium was replaced every other day. Cells were counted and scored for the presence of neurites after 1 (D1). 3 (D3), or 5 days (D5). Bar, 100 µm.



In view of previous reports suggesting that EGF induced a rapid decrease in its own cell surface receptors(19) , we examined the levels of EGFR phosphorylation as a function of time in cells stimulated with EGF in the presence and absence of staurosporine. As shown in Fig. 3A and Fig. 4, A and C, cells pretreated with staurosporine for 16 h clearly show more extensive phosphorylation of the EGFR (when exposed to EGF), as detected with anti-phosphotyrosine antibodies and I-protein A, than cells treated with EGF alone. The identification of the 170-kDa band as EGFR was confirmed by stripping and reprobing the blots with anti-EGFR antibody (Fig. 4, B and D). The blots in Fig. 3also revealed an increased level of phosphorylated proteins in the molecular weight range expected for the two isoforms of MAPK (pp42 and pp44), which is known to correspond to an increased activity of these enzymes(11, 36) . This was confirmed by stripping the blot and reprobing with antibodies specific for these entities. As shown in the lowerpanel of Fig. 3, the levels of phosphorylated MAPK1 and -2 (indicated by the shift in mobility in the lanes corresponding to 2 and 10 min (leftside) and 2, 10, 30, and 60 min (rightside)) are indeed prolonged in the cells pretreated with staurosporine and induced by EGF (both the phosphorylated and dephosphorylated forms are clearly visible in the later time points of the staurosporine plus EGF samples). This latter profile is virtually identical to that seen for NGF and basic FGF in PC12 cells, under conditions that produce full neurite outgrowth(37) . It is important to point out that, at the concentration used, staurosporine by itself had no effect on the basal level of tyrosine phosphorylation in PC12 cells and on the activity of MAPK1 and MAPK2, as shown in Fig. 3B.


Figure 3: Time course of protein tyrosine phosphorylation (A) and of MAPKs activity (B) induced by EGF in the absence and presence of staurosporine. A, after preincubation without (leftpanel) or with (rightpanel) 10 nM staurosporine for 16 h, PC12 cells were left untreated (0) or treated with EGF (10 ng/ml) for 2, 10, 30, 60, 120, and 240 min at 37 °C as indicated. 100 µg of protein from each whole cell lysate was analyzed by 7.5% SDS-PAGE followed by immunoblotting with a polyclonal anti-phosphotyrosine antibody, detection with I-protein A, and autoradiography. Specific proteins are indicated by arrowheads. The migration of molecular mass standards (in kDa) is shown on the left and on the right for each gel. B, the anti-phosphotyrosine blots were stripped and reprobed with anti-ERK-1 antiserum. The antiserum recognizes both p44 and p42 as indicated by arrowheads.




Figure 4: Time course of EGF receptor tyrosine phosphorylation (A, C, and E) and level of EGF receptor (B, D, and F) in PC12 cells in the absence and presence of staurosporine. After preincubation without (A, B) or with (C, D) 10 nM staurosporine for 16 h, PC12 were left untreated (lanes0) or treated with EGF (10 ng/ml) for 2, 10, 30, 60, 120, and 240 min at 37 °C as indicated. A and C, 100 µg of protein from each whole cell lysate was analyzed by 7.5% SDS-PAGE followed by immunoblotting with the antiphosphotyrosine antiserum. B and D, the anti-phosphotyrosine blots were stripped and reprobed with an anti-EGFR monoclonal antibody C13. E and F, after preincubation without or with staurosporine for 16 h, PC12 cells were left untreated or treated with EGF for 30 min, as indicated, lysed, and immunoprecipitated with monoclonal anti-rat EGFR antibody. Immunoprecipitates were separated by SDS-PAGE, transferred to Immobilon P membranes, and immunoblotted with a polyclonal anti-phosphotyrosine antibody (E) and, after stripping, reprobed with a monoclonal anti-EGFR antibody generated against a peptide corresponding to the submembrane domain of the human EGFR (F). The phosphorylation of p170, corresponding to the EGFR, is indicated by arrowheads.



To confirm that the increased phosphorylation of the 170-kDa species corresponds to the EGFR, immunoprecipitation and Western blot analyses were performed. As shown in Fig. 4, E and F, after immunoprecipitation with anti-EGFR antibody and anti-phosphotyrosine antibody Western blotting, cells treated with EGF (pretreated with staurosporine) for 30 min show a more heavily phosphorylated EGFR than cells treated with EGF alone. After stripping and reprobing the blot with anti-EGFR antibody, it is evident that more EGFR can be immunoprecipitated in the cells treated with either both agents or staurosporine alone (Fig. 4F). Despite the higher level of EGFR present in PC12 cells following their pretreatment with staurosporine for 16 h, no tyrosine phosphorylation of this receptor was observed until EGF was added to the culture medium. It appears that the up-regulation of the level of EGFR in cells pretreated with staurosporine is evident, even if in a more qualitative manner, in the anti-EGFR Western blot of total cell extracts of PC12 cells when comparing the first three left lanes of Fig. 4D (cells treated with staurosporine for 16 h) to the corresponding lanes of Fig. 4B (untreated cells).

As the up-regulation of EGFR by the prolonged treatment with staurosporine cannot rule out a possible direct effect of staurosporine on the phosphorylation of EGFR, experiments were performed to address this issue. Upon simultaneous addition of EGF and staurosporine, a modestly increased phosphorylation of EGFR and MAPKs was observed when compared with the addition of EGF alone up to 30 min (Fig. 5). This transient stimulatory effect of staurosporine on EGF-induced protein tyrosine phosphorylation is likely due to a direct inhibition of phosphorylation of EGFR at Ser-1046/Ser-1047 (19) or on other phosphorylation/dephosphorylation events that regulate EGFR activity. Thus, they may make some contribution to the induced EGF responsiveness.


Figure 5: Time course of protein tyrosine phosphorylation induced by EGF with or without the simultaneous addition of staurosporine. PC12 cells were left untreated (0) or treated with EGF (10 ng/ml) for 2, 10, 30, 60, 120, and 240 min at 37 °C without (leftpanel) or with the addition of 10 nM staurosporine (rightpanel). 100 µg of protein from each whole cell lysate was analyzed by 7.5% SDS-PAGE followed by immunoblotting with a polyclonal anti-phosphotyrosine antibody, detection with I-protein A, and autoradiography. Specific proteins are indicated by arrowheads. The migration of molecular mass standards (in kDa) is shown on the left and on the right for each gel.



The clear demonstration that PC12 cells treated with staurosporine show a sustained response to EGF, including a prolonged level of phosphorylated receptor at the plasma membrane, suggests an altered rate of synthesis or degradation (down-regulation). To test whether staurosporine effected the expression of EGFR, PC12 cells were treated for 2, 4, 8, and 16 h with 10 nM drug and 20 µg of total RNA isolated from each. Following separation on 1% agarose, blots were probed at 65 °C with a riboprobe corresponding to the extracellular domain of PC12 EGFR. As shown in Fig. 6, staurosporine is a potent inducer of EGFR gene expression in these cells. The effect appears to peak at 8 h but remains high at 16 h. Translation of this increased level of mRNA would presumably increase the number of plasma membrane EGFR, similar to cells transfected with an exogenous EGFR gene (18) , and account for the increased tyrosine phosphorylation signals induced by the added EGF ( Fig. 3and Fig. 4). It is unclear whether the mechanism by which staurosporine effects the level of EGFR gene transcription is related to kinase inhibition or to some other as yet undefined activity. However, similar observations have been made with human tumor necrosis factor receptors on myeloid and epithelial cells (38) , suggesting the phenomenon may be a more general one.


Figure 6: Northern blot analysis of the effect of staurosporine on the expression of EGFR mRNA level. PC12 cells were cultured in complete medium in the absence or in the presence of 10 nM staurosporine for 0, 2, 4, 8, and 16 h as indicated. At the end of this period, total RNA was extracted from cells, and 20 µg of each sample was fractionated on a 1% formaldehyde-agarose gel. The blot was first probed at 65 °C with a rat EGFR riboprobe; it was then stripped and reprobed at 42 °C with a CHOb probe to normalize RNA loading differences.



These findings clearly establish that the endogenous EGFR of PC12 cells is capable of inducing neurite outgrowth, effectively eliminating the possibility that it fails to do so in wild type PC12 cells because of some abnormality in function. These results further suggest that any substantial increase in plasma membrane levels of EGFR will allow for EGF-induced differentiation in these cells, in this case provided by staurosporine-induced increases in expression or inhibition of down-regulation. In contrast to the situation expected for stably transfected cells(18) , the sustained expression of EGFR is eventually overcome, i.e. the up-regulation by staurosporine is not permanent, and neurite outgrowth begins to wane by day 5. This provides further proof that the effect observed is related to the number of EGFR and not to any changes in the specificity of its receptor kinase.

It also suggests that the magnitude of the signal induced and the length of time that threshold levels of phosphorylation can be maintained are directly related to the number of cell surface receptors. There is greater than an order of magnitude more EGFR than TrkA or FGF receptor on native PC12 cells (^2)(although the relative amounts of each receptor type can vary, particularly in different PC12 cell subspecies), which might suggest that the EGFR tyrosine kinase has a lower specificity for substrates of the Ras pathway. Its response is essentially identical to that of the NGF receptor (TrkA) in the first 3 min(17, 18) , which may represent the maximal magnitude of this response, and more EGFR may be required to achieve this. However, in view of the rapid decay of the signal, it is equally likely that there is no significant difference in substrate selectivity or affinity of the EGFR for activating Ras, thus suggesting that inherently EGF is potentially as good a neurotrophic factor as NGF and FGF. It is interesting in this regard that it has been reported to affect selected neurons in the central nervous system(39, 40) , although it is not reported to act on neuronal targets in the peripheral nervous system.

The importance of the duration of the MAPK signal in PC12 cells may be related to both locational and temporal aspects of the response. Clearly, the prolonged response induced by NGF and basic FGF causes not only the activation of MAPK but also its translocation to the nucleus (17) . This may be fundamental to the initiation of the neurotrophic response. It is instructional to recall that the initial response of PC12 cells to all of these factors is a mitotic one(41) , i.e. there is apparently a protein(s) that must be induced before the neurotrophic response can be manifested. This clearly requires new protein synthesis, suggesting that the signal induced by a potential neurotrophic factor must sustain the MAPK signal long enough to produce an initial round of transcription (expression of the set of immediate early response genes and their products(42) ). Cells that have been primed, i.e. exposed previously to NGF(5) , or variants that can respond essentially immediately to growth factor (43, 44) do not have this temporal restriction, presumably because they have previously synthesized the required regulatory protein(s). Although this putative entity has yet to be described, its behavior is consistent with that expected for a mitotic suppressor(45) . It might also be noted that the transcriptional regulation controlled by MAPK is sufficiently extensive that it leads to a clearly defined phenotype following 2-6 h of stimulation by NGF or FGF that is apparently the signature of the differentiated cell and is lacking following EGF treatment(35) .


FOOTNOTES

*
This work was supported by Research Grants AG09735 from the National Institutes of Health and BE-41N from the American Cancer Society. 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.

§
To whom correspondence should be addressed: Dept. of Biological Chemistry, California College of Medicine, Medical Science Bldg. I, Irvine, CA 92717-1700. Tel.: 714-824-6236; Fax: 714-824-8036; rablab{at}uci.edu.

(^1)
The abbreviations used are: NGF, nerve growth factor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; MAPK (or ERK), mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis.

(^2)
R. A. Bradshaw and K. Seedorf, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Gordon Gill, University of California at San Diego, and Maureen O'Connor-McCourt, National Research Council Canada, for generously providing anti-EGFR antibodies. Also, we thank Drs. Gill, Albert Stewart, Didier Thomas, and Yvonne Wu for helpful discussions. The expert assistance of Gale Trudeau in the preparation of this manuscript is appreciated.

Note Added in Proof-After this manuscript was submitted, Isono et al. (46) reported similar effects of K-252a, a compound related to staurosporine, on the response of PC12 cells to EGF.


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