(Received for publication, October 8, 1996, and in revised form, February 17, 1997)
From the Section on Growth Factors, NICHD, National Institutes of Health, Bethesda, Maryland 20892 and § Center of Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892
Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor receptors during the differentiation of PC12 cells. This process is characterized by a progressive decrease in epidermal growth factor (EGF) receptor level measured by 125I-EGF binding, tyrosine phosphorylation, and Western blotting. Treatment of the cells with NGF for 5 days produces a 95% reduction in the amount of [35S]methionine-labeled EGF receptors. This down-regulation does not occur in PC12nnr5 cells, which lack the p140trk NGF receptor. However, in PC12nnr5 cells stably transfected with p140trk, the NGF-induced heterologous down-regulation of EGF receptors is reconstituted in part. NGF-induced heterologous down-regulation, but not EGF-induced homologous down-regulation of EGF receptors, is blocked in Ras- and Src-dominant-negative PC12 cells. Treatment with either pituitary adenylate cyclase-activating peptide (PACAP) or staurosporine stimulates neurite outgrowth in PC12 cell variants, but neither induces down-regulation of EGF receptors. NGF treatment of PC12 cells in suspension induces down-regulation of EGF receptors in the absence of neurite outgrowth. These results strongly suggest a p140trk-, Ras- and Src-dependent mechanism of NGF-induced down-regulation of EGF receptors and separate this process from NGF-induced neurite outgrowth in PC12 cells.
PC12 cells, derived from a rat pheochromocytoma, have been
extensively used as a model of neuronal differentiation (1), because
the cells acquire the phenotype of sympathetic neurons and stop
dividing in response to nerve growth factor
(NGF)1 (2). In PC12 cells, NGF interacts
with two distinct plasma membrane receptor proteins:
p75NGFR, a cysteine-rich glycoprotein having a relatively
low affinity for NGF (3), and p140trk, a receptor tyrosine
kinase activated by NGF, which binds NGF with high affinity and
mediates many of the biological activities of this neurotrophin (4).
The binding of NGF to the p140trk receptor stimulates rapid
tyrosine autophosphorylation of the receptor and activation of several
signal-transducing proteins (4). Activated p140trk receptors
bind to and tyrosine phosphorylate the signaling substrates phospholipase C1, phosphatidylinositol-3 kinase, and Src homologous collagen protein (4), resulting in their activation. The latter stimulates Ras activity which subsequently activates a series of
serine/threonine kinases including B-Raf, myelin basic protein kinase
kinase, the Erks, and p90rsk (4). This Ras-Erk pathway plays a
major role in the activation of transcriptional events by NGF and in
NGF-induced neuronal differentiation (5), as illustrated by inhibition
of NGF actions upon expression of dominant-negative Ha-Ras (Asn-17)
proteins (6, 7). In addition a role for pp60c-src
in NGF actions has been suggested (5) by experiments involving the
expression of a dominant-interfering kinase-inactive Src (8).
PC12 cells also express plasma membrane receptors for epidermal growth factor (EGF) (9, 10), a mild mitogen for these cells (9). PC12 cells treated with NGF exhibit an attenuated response to EGF (9) because EGF receptors are down-regulated (9, 11). The cellular mechanisms that mediate NGF-induced EGF receptor down-regulation are unknown and represent an interesting question in neuronal development. The existence of functional receptors for both NGF and EGF on PC12 cells and the recent advances in understanding the signaling pathways activated by these receptors make these cells a useful model for the study of cross-regulation between NGF and EGF receptors during differentiation.
In this study, we have used a number of PC12 variant cell lines with different levels of p140trk (12, 13) and EGF receptor expression and dominant-negative Ras (7, 14, 15) or Src (5, 6, 8) PC12 transfectants in an attempt to elucidate the molecular mechanisms of the receptor cross-talk required by NGF to down-regulate EGF receptors. Our findings implicate the involvement of p140trk-, Ras-, and Src-dependent signaling pathways in NGF-induced down-regulation of EGF receptors while demonstrating an independence of this cellular process from NGF-induced neurite outgrowth.
Mouse NGF, EGF, bovine brain-derived acidic FGF
and basic FGF, and rat collagen type II were purchased from
Collaborative Biochemicals (Bedford, MA). Dexamethasone,
poly-L-lysine, and myelin basic protein were purchased from
Sigma. K-252a and staurosporine were prepared at Kyowa Hakko Kogyo,
Ltd. (Tokyo, Japan). Pituitary adenylate cyclase-activating polypeptide
(rat PACAP-38) and the PACAP receptor antagonist PACAP (6-38) were
purchased from Peninsula Laboratories (Belmont, CA). Heparin was
obtained from Hepar (Franklin, OH). The monoclonal anti-phosphotyrosine
(4G10) and anti-EGF receptor (6F1) antibodies were purchased from
Upstate Biotechnology Inc. (Lake Placid, NY) and Medical and Biological
Laboratories, Co. Ltd (Nagoya, Japan), respectively. The polyclonal
anti-trkA (C14), anti-Src, anti-Ras, and anti-Erk1 antibodies were
products of Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The
polyclonal anti-EGF receptor antibody 1210 was raised against a
12-amino acid peptide derived from the intracellular domain of the EGF receptor (16). Rabbit anti-neurofilament M C-terminal polyclonal antibody was purchased from Chemicon International Inc. (Temecula, CA).
Murine 125I-EGF (specific activity 180 µCi/µg) and
125I-NGF (specific activity 70.6 µCi/µg) were obtained
from DuPont NEN. [3H]Thymidine (specific activity, 85 Ci/mmol) and [-32P]ATP were purchased from Amersham
Corp. 125I-FGF was prepared as described previously
(17).
PC12 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.) supplemented with 7% fetal bovine serum, 7% horse serum, 100 µg/ml streptomycin, and 100 units/ml penicillin, all from Life Technologies, Inc. (11). In all experiments involving extended treatment with NGF, the medium was changed, and fresh NGF was added every other day. PC12nnr5 cells were grown in collagen-coated tissue culture dishes in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% horse serum and 5% fetal bovine serum (13). 6.24 cells, a clone of PC12 overexpressing human p140trk, were grown under the same conditions as PC12 cells in the presence of 200 µg/ml G418 (Life Technologies, Inc.) (12). During the down-regulation experiments, G418 was removed from the medium. The PC12 cell variants, GSrasDN6 expressing a dominant-negative mutant ras gene (15) under the transcriptional control of the mouse mammary tumor virus promoter, M-M17-26 expressing the Ha-ras Asn-17 gene under the transcriptional control of the mouse metallothionein-I promoter (7), and srcDN2 expressing the K295R mutant (kinase dead) form of chicken Src under the control of the cytomegalovirus promoter (8) were grown, as were PC12 cells, in DMEM supplemented with 10% fetal bovine serum and 5% horse serum. In a series of experiments involving dexamethasone treatment, the cells were placed in charcoal-treated serum 24 h before stimulation with 500 nM dexamethasone. To grow cells in suspension, PC12 cells were dispersed in 50 ml of complete medium at a density of 1 × 105 cells/ml in a 250-ml polycarbonate spinner flask (Corning) (18). The suspension was gently shaken at 37 °C on a rotary shaker in an incubator.
Binding AssaysRadioreceptor assays were carried out as
described previously (11). The cells were grown on collagen (200 µg/ml)- and polylysine (10 µg/ml)-coated six-well plates (Costar).
Cell culture monolayers were washed and equilibrated with fresh,
serum-supplemented DMEM for 1 h. Total binding of growth factors
was measured by adding saturating concentrations of
125I-EGF (0.5 × 106 cpm/ml) or 50-100
pM 125I-NGF (to measure high affinity
p140trk receptors). Nonspecific binding was evaluated by adding
a 100-fold excess of unlabeled growth factors to sister cultures and
was typically 5-10% and 20-30% of total binding for EGF and NGF,
respectively. The incubation, unless otherwise stated, was carried out
at 37 °C for 45 min, then the incubation buffer was removed, and the
cells were washed with ice-cold phosphate-buffered saline (138 mM NaCl, 2.7 mM KCl, 8 mM
Na2HPO4, 1.5 mM
KH2PO4). The cell monolayer was solubilized
with 1 ml of 1 N NaOH overnight at room temperature. The
cell-associated radioactivity was counted in a -counter; a portion
of the solubilized mixture was used for protein determination. Data
points represent specific binding values and are the means of
quadruplicate samples minus the nonspecific binding, expressed as
counts/min/µg of protein (EGF) or counts/min/mg of protein (NGF) ± S.D. Binding experiments with cells grown in suspension were performed
for 45 min in suspension, using 3 × 106 cells/ml of
medium with 125I-EGF (1 × 106 cpm/ml) in
the presence or absence of 10 µg/ml unlabeled EGF. The separation of
free 125I-EGF was done by centrifuging the cells three
times each for 10 min at 4 °C. Binding studies using plasma membrane
preparations (11) were performed similarly at 4 °C, except that free
and membrane-bound EGF were separated by Microfuge centrifugation (12,000 × g) at 4 °C using phosphate-buffered
saline buffer containing 0.1% bovine serum albumin, pH 7.4. Down-regulation was performed by incubation of the cultures at 37 °C
with either NGF (2 nM) or EGF (1 nM) for the
period of time indicated. The monolayers were washed to remove the
unbound growth factors, incubated in fresh DMEM for 2 h at
37 °C to allow internalization of remaining growth factor-receptor
complexes (10, 11), and after two additional washes with medium, the
extent of receptor down-regulation was estimated by the amount of
125I-growth factor associated with the cell. FGF binding
was assessed as described above with the exception that the cultures
were used 24 h after plating, and the radioreceptor assay was
performed in 200 µl of binding buffer (DMEM containing 10 µl/ml
heparin, 0.2% bovine serum albumin, 25 mM HEPES, pH 7.4)
with 0.4 ng of either 125I-labeled acidic FGF or
125I-labeled basic FGF in the presence or absence of 50 ng
of the respective unlabeled growth factor, as described previously
(17). Binding was terminated with two washings with binding buffer
followed by a third wash for 5 min with binding buffer at pH 7.5 or pH 4.0 for low and high affinity FGF binding, respectively (17).
For iodination of cell surface proteins, 3 × 106 cells grown on 10-cm collagen- and polylysine-coated tissue culture dishes were incubated with 1 mCi of Na125I and 0.2 mg/ml lactoperoxidase (19). In other experiments, PC12 cell cultures were incubated for 18 h in methionine-free DMEM containing 100 µCi/ml [35S]methionine/cysteine (Translabel; ICN, Plainview, NY). For immunoprecipitation of EGF receptors, the cell monolayers were washed twice with DMEM and solubilized for 30 min at 4 °C in radioimmune precipitation buffer (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin) (11). Insoluble material was removed by centrifugation, and solubilized material was subjected to immunoprecipitation using anti-EGF receptor antibody.
Immunoprecipitation and ImmunoblottingCells were plated in
10-cm tissue culture dishes 1 day prior to the experiment. Following
treatment with growth factors, cells were washed twice with ice-cold
Tris-buffered saline (20 mM Tris-HCl, pH 8.0, 137 mM NaCl) and subjected to lysis in 1 ml of 1% Nonidet P-40
in lysis buffer (20 mM Tris-HCl, 137 mM NaCl,
0.5 mM EDTA, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 0.15 unit/ml aprotinin, 20 mM leupeptin, 1 mM sodium vanadate) at 4 °C
for 20 min. Insoluble material was removed by centrifugation for 10 min
at 12,000 × g, and lysates were subjected to
immunoprecipitation with anti-trkA or anti-Erk antibodies for 2 h
at 4 °C with continuous agitation, followed by an additional 2-h
incubation with protein A-Sepharose. Precipitates were washed three
times with lysis buffer and once with water, then boiled for 5 min in
SDS sample buffer (0.06 mM Tris-HCl, pH 6.8, 12.5%
glycerol, 1.25% SDS, 5% -mercaptoethanol, 0.002% bromphenol
blue), and subjected to SDS-PAGE on 7.5% polyacrylamide gels, followed
by transfer to nitrocellulose. Blots were probed overnight at 4 °C
with the primary antibodies and analyzed using an enhanced or Tropix
chemiluminescence system and horseradish peroxidase-coupled secondary
antibodies and quantitated by densitometry.
EGF-dependent tyrosine kinase
activity was assayed in both whole cells and cell-free membrane
fractions. PC12 cells were labeled for 4 h with
[32P]orthophosphate (0.25 mCi/ml) in phosphate-free DMEM
(16). For membrane experiments, 200 µg of PC12 cell membranes were
preincubated with EGF (10 ng/ml) for 15 min at 37 °C in a reaction
buffer containing 20 mM HEPES, pH 7.2, 0.5 mM
MgCl2, 3 mM MnCl2, and 50 µM Na3VO4 in a final volume of 50 µl (13). The reaction was initiated by the addition of 2 µCi of
[-32P]ATP in 50 µM unlabeled ATP for 5 min at 4 °C. The reaction was terminated by the addition of SDS
sample buffer, and the samples were analyzed by SDS-PAGE and
autoradiography (11). In other experiments, the kinase activity of EGF
receptors eluted with N-acetylglucosamine (300 mM) from wheat germ lectin-Sepharose chromatography (11)
was evaluated in a similar way.
Washed protein A-agarose pellets
containing immunoprecipitated Erk1 were suspended in 30 µl of kinase
assay buffer (7.5 mM HEPES, pH 7.5, 10 mM
MgCl2, 1 mM dithiothreitol, 2.5 µM protein kinase A inhibitor peptide PKI (6-22)-amide,
225 µM cold ATP, 25 µCi of [-32P]ATP,
500 µg/ml MBP as kinase substrate) and incubated for 30 min at room
temperature. The reaction was terminated by the addition of 30 µl of
2 × SDS sample buffer. Samples were heated for 5 min in a boiling
water bath then electrophoresed through a 4-20% Tris-glycine gradient
gel (NOVEX). Following electrophoretic resolution, radiolabeled proteins were transferred to polyvinylidene fluoride membranes. Phosphorylated MBP was visualized by exposure of the membranes to
Biomax MR film (Eastman Kodak Co.). MBP-associated radioactivity was
quantitated directly from the polyvinylidene fluoride membranes using
an AMBIS radioanalytic imaging system and confirmed by
scintillation counting of the excised bands.
To evaluate neurite outgrowth, cells were plated at low density (1,000-5,000 cells/dish) on collagen- and polylysine-coated tissue culture dishes in the appropriate medium. The cells were treated for different periods of time with the indicated reagents, examined by light microscopy in an inverted Nikon-Diaphot microscope, and photographed at × 320 magnification.
The level of
125I-EGF binding during NGF-induced differentiation of PC12
cells was examined (Fig. 1). Treatment of the cells with
50 ng/ml NGF progressively reduced the level of 125I-EGF
binding; 50% reduction was seen after 3 days of treatment. In
wild-type PC12 cells, this decrease in binding represents a decrease in
EGF receptor level (Fig. 1, bottom) and accompanies the
NGF-induced elongation of neurites (Fig. 1, top). The
neuropeptide PACAP induces similar, but somewhat less robust neurite
outgrowth in PC12 cells, but does not affect the binding of
125I-EGF (Fig. 2A).
Staurosporine, a microbial protein kinase inhibitor, also induces rapid
neurite outgrowth in PC12 cells, but, in contrast to NGF, increases
125I-EGF binding up to 3-fold (Fig. 2B). By way
of comparison, NGF-induced differentiation of PC12 cells does not
affect the level of either high or low affinity receptors for acidic or
basic FGF (Table I) or of receptors for insulin-like
growth factor I (20).
|
NGF-induced down-regulation of EGF receptors in PC12 cells can also be
measured in isolated plasma membrane fractions derived from cells
treated with NGF (Fig. 3A). Iodination of EGF
receptors on PC12 cell membranes with lactoperoxidase and
Na125I in control and 5-day NGF-treated cells, followed by
immunoprecipitation of the labeled EGF receptors indicated a 98%
decrease in receptor level (Fig. 3B). Another criterion of
NGF-induced, down-regulation of EGF receptors would be a decrease in
EGF-stimulated tyrosine kinase activity of EGF receptors. In both whole
cells (Fig. 3C) and membrane fractions (Fig. 3D),
phosphorylation of EGF receptors was very low or absent after 7 days of
NGF treatment. When the levels of
[35S]methionine-labeled, immunoprecipitated EGF receptors
in control and 7-day NGF-treated PC12 cells were compared (Fig.
3E), 60 and 95% decreases in the level of 170- and 150-kDa
EGF receptor proteins, respectively, were seen.
NGF-induced Down-regulation of EGF Receptors Is p140trk-dependent
PC12 cells express two types of receptors for NGF: the high affinity receptor p140trk and the low affinity receptor p75NGFR (3). In PC12nnr5 cells, a PC12 variant that expresses p75NGFR, but very little p140trk (13), NGF does not induce neurite outgrowth. When human p140trk is transfected into PC12nnr5 cells, they grow neurites in response to NGF stimulation (21). Also, overexpression of p140trk in wild-type PC12 cells accelerated NGF-induced neurite outgrowth in the 6.24 PC12 cell variant (12).
To determine if NGF-induced down-regulation of EGF receptors is
mediated by p140trk receptors, we first estimated the levels of
NGF and EGF receptors in these several PC12 cell variants (Fig.
4). Indeed, compared with wild-type PC12 cells, PC12nnr5
cells express very low levels of p140trk receptors, but had
63% of the EGF receptors seen in the wild-type PC12 cells, while the
6.24 cell variant expressed 240 and 205% more p140trk and EGF
receptors, respectively, than did the wild-type. The level of EGF
receptors in PC12nnr5 cells was not altered, even after 7 days of NGF
treatment as evaluated by binding (Fig. 5). However, the
EGF receptors in these cells could be rapidly homologously down-regulated by exposure of the cells to EGF. This is a reversible process, as indicated by the reappearance of cell surface binding upon
removal of EGF (Fig. 5).
PC12nnr5 cells were stably transfected with human p140trk as recently described (21). Out of 120 clones generated, a dozen were selected based on their expression of p140trk and EGF receptors and the ability of NGF treatment to down-regulate the EGF receptors. As indicated in Table II, four clones were further characterized. Clone 42 is a p140trk overexpressor, clones 61 and 106 have levels of p140trk similar to those in PC12 cells, and clone 50 expresses much lower levels of p140trk (Table II). These binding data are supported by the Western blots presented in Fig. 6, indicating different degrees of EGF receptor down-regulation in the four clones. This figure also shows that the transfected trk receptors are functional in that they mediate the stimulation of Erk phosphorylation by 50 ng/ml NGF (Fig. 6). The NGF-induced down-regulation of EGF receptors was measured in all four clones. No direct correlation was found between NGF-induced neurite outgrowth and NGF-induced down-regulation of EGF receptors (Table II). NGF does not down-regulate EGF receptors in PC12nnr5 cells, but does so (30-60% down-regulation) after introduction of p140trk into these cells (Table II). Therefore, it seems likely that NGF-induced down-regulation is mediated by p140trk. Support for this suggestion was also provided by experiments with K-252a (Fig. 7). K-252a, a specific inhibitor of NGF-induced p140trk tyrosine kinase activity in whole PC12 cells (22), prevented the down-regulation of EGF receptors by NGF in both parental PC12 cells (Fig. 7A) and 6.24 PC12 p140trk-overexpressing cells (Fig. 7B). Although the NGF-induced neurite outgrowth in 6.24 cells is accelerated as it is in PC12nnr5-p140trk.42 cells, the time course of NGF-induced down-regulation of EGF receptors is similar to that in wild-type PC12 cells (Fig. 1), reaching 50% decrease in receptor levels in about 48-72 h (Fig. 7B).
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NGF-induced Down-regulation of EGF Receptors is Ras-dependent
To test whether NGF-induced
down-regulation of EGF receptors is dependent upon Ras activation, two
PC12 cell variants, transiently (GSrasDN6) or stably (M-M17-26)
overexpressing the dominant-negative mutant RasN17 were
employed. As shown in Fig. 4, these variants express different levels
of p140trk and EGF receptors. Expression of the
dominant-negative Ras protein is induced in GSrasDN6 and increased in
M-M17-26 by treatment with dexamethasone (14, 15). To determine whether
the dominant-negative Ras protein affects the stimulation of Erks under
the conditions in which NGF-induced down-regulation was measured, we
examined NGF- or EGF-induced phosphorylation of MBP by
immunoprecipitated Erks in GSrasDN6 cells and NGF-induced
phosphorylation of MBP by immunoprecipitated Erks in M-M17-26 cells
compared with wild-type PC12 cells (Fig. 8C).
In GSrasDN6 cells not treated with dexamethasone, NGF and EGF induced a
12- and 11-fold increase in Erk1 activity, respectively, measured by
the phosphorylation of myelin basic protein (Fig. 8C). In
dexamethasone-treated GSrasDN6 cells, the NGF- or EGF-induced increases
in Erk1 activity were almost completely inhibited (Fig. 8C).
Under identical conditions, a 25% reduction in 125I-EGF
binding could be measured after 48 h of NGF treatment (Fig. 8A) or 4-5 days (data not shown) in GSrasDN6 cells not
treated with dexamethasone (Fig. 8A). However, a complete
homologous down-regulation of EGF receptors could be observed (Fig.
8A). Upon dexamethasone treatment, the small NGF-induced
down-regulation effect was blocked, but not the EGF-induced homologous
down-regulation of EGF receptors (Fig. 8A).
In M-M17-26 cells in which the dominant-negative RasN17 is stably overexpressed, neither NGF nor EGF stimulated Erk1-induced MBP phosphorylation (Fig. 8C). In these cells, NGF-induced heterologous down-regulation of EGF receptors is completely blocked (Fig. 8B), but the EGF-induced homologous down-regulation (100 in 1 h) is unaffected (data not shown).
NGF-induced Down-regulation of EGF Receptors is Src-dependentThe SrcDN2 PC12 cell variant,
overexpressing a kinase-inactive, dominant-negative Src was treated
with NGF and 125I-EGF binding was measured (Fig.
9). These cells express 46% of the p140trk and
86% of the EGF receptors seen in wild-type PC12 cells (Fig. 4). NGF
treatment for 4 days did not induce the down-regulation of EGF
receptors (Fig. 9) typical of wild-type PC12 cells. As expected,
Western blotting analysis also did not show any change in the level of
EGF receptors in SrcDN2 cells treated for 5 days with 50 ng/ml NGF
compared with untreated cells. However, treatment of the cells with 50 ng/ml EGF for 1 h at 37 °C induced 86% homologous down-regulation of the EGF receptors (data not shown).
NGF-induced Down-regulation of EGF Receptors Is Independent of Neurite Outgrowth
In view of the observation that in wild-type
PC12 cells, the NGF-induced down-regulation of EGF receptors is
concomitant with NGF-induced neurite outgrowth, we cultured PC12 cells
for 7 days either in monolayer or suspension (18) cultures in the
presence or absence of 50 ng/ml NGF. In monolayer cultures the cells
grow neurites in the presence of NGF; in suspension they aggregate, but
do not grow neurites (18). Thereafter the cells were harvested and
analyzed by Western blotting with either anti-EGF receptor antibody to
measure EGF receptor down-regulation or anti-neurofilament-M antibody
to measure the efficiency of the NGF treatment of the cells. As seen in
Fig. 10, NGF induced down-regulation of EGF receptors in suspension cultures in the absence of neurite outgrowth after 7 or
10 days of NGF treatment. Binding experiments with 125I-EGF
indicated a decrease of 68 and 82% in EGF receptors in suspension cultures treated for 7 and 10 days with NGF, respectively, compared with 85% decrease in EGF receptors in monolayer cultures.
Both acute and chronic heterologous regulation of EGF receptors has been demonstrated in many cells. This regulation process is characterized by a change in either the affinity or the number of EGF receptors. For example, a decreased affinity of EGF receptors was measured upon treatment of cells with platelet-derived growth factor (23), vasopressin (24), interleukin 1 (25), and phorbol esters (26), and a decrease in the number of EGF receptors was observed in response to thyroid hormone (27), norepinephrine (28), nerve growth factor (11), or after infection with adenovirus (29, 30) or Rous sarcoma virus (31). In a few cases, the heterologous up-regulation of EGF receptors has been seen. An increased number of EGF receptors is evident in 3T3-L1 fibroblasts treated with 3-deazaadenosine (32), in teratocarcinoma cells treated with retinoic acid (33), or upon oncogenic transformation of carcinoma cells (34). The cellular signaling pathways utilized by G protein-coupled receptors, tyrosine kinase receptors, viruses, or drugs to regulate EGF receptors are as yet poorly characterized.
PC12 cells possess receptors for EGF, FGF, and NGF and have been used to investigate the process of heterologous down-regulation of EGF receptors by NGF. In these cells, both an acute (10 min) (35, 36), as well as a chronic (several days) (10, 11) down-regulation of EGF receptors in response to NGF has been reported. This biphasic down-regulation process was also observed upon NGF-induced down-regulation of c-neu receptors by NGF in PC12 cells (16). The present data emphasize the three major characteristics of the NGF-induced, chronic down-regulation of EGF receptors in PC12 cells: (a) a decrease in receptor level on the cell surface; (b) a disappearance of EGF-stimulated, receptor-mediated tyrosine kinase activity; and (c) a decrease in receptor synthesis.
Mechanisms have been proposed for the acute NGF-induced down-regulation of EGF receptors (35, 36). Acute regulation of EGF receptors has been attributed to activation of either protein kinase C, which phosphorylates threonine 654 of the EGF receptor (37), or of calmodulin-dependent protein kinase II, which phosphorylates serine 1046/1047 (38), or of other unknown kinases (39, 40), which might be also the case for PC12 cells. On the other hand, the signaling pathway(s) utilized by NGF for the chronic down-regulation of EGF receptors has not been addressed. The chronic, NGF-induced down-regulation of EGF receptors in PC12 cells is definitely different in mechanism from the acute down-regulation. It is selectively induced by p140trk receptors, but not by G protein-coupled PACAP receptors or drug (staurosporine)-induced neurite outgrowth. This process is not due to a change in receptor affinity (10, 11), receptor processing, or receptor internalization (11, 16). The lack of NGF-induced heterologous down-regulation of EGF receptors in PC12nnr5 cells, the partial reconstitution of the heterologous down-regulation in PC12nnr5-p140trk transfectants, and the ability of K-252a to block NGF-induced EGF receptor down-regulation support the concept that this effect is mainly mediated by the high affinity NGF receptor, p140trk, and not by the low affinity NGF receptor, p75NGFR. This conclusion is also supported by the ability of both Src and Ras dominant-negative forms to block this cellular process, since these two signal transduction pathways are initiated by binding of NGF to p140trk receptors, but not by binding of NGF to p75NGFR (5).
Src has been implicated, in PC12 cells, in calcium-stimulated signaling events (5, 6, 8) as well as in tyrosine phosphorylation of cytoskeletal proteins (41), either of which might also be involved in NGF-induced down-regulation of EGF receptors. Alternatively, since expression of the dominant-negative Src blocked the tyrosine phosphorylation of both Src homologous collagen protein and mitogen-activated protein kinases (5, 6), the inhibition of NGF-induced down-regulation of EGF receptors might simply reflect the blockade of the Ras signaling pathway. Because Fyn, Yes, and other tyrosine kinase members of the Src family could also be blocked by overexpression of the kinase-inactive Src (8), there is a possibility that these kinases, as well, may be involved in NGF-induced down-regulation of the EGF receptors.
One possible explanation of our findings is that Ras-Erk activity is an obligatory element in p140trk-mediated pathway required for chronic NGF-induced, down-regulation of EGF receptors, affecting an event such as phosphorylation of protein(s) involved in the transcription, translation, and/or post-translational modification of EGF receptors.
Alternatively, it is possible that Ras suppression of NGF-induced neurite outgrowth indirectly blocked NGF-induced down-regulation of EGF receptors. This possibility seems unlikely, however, because our data suggest a lack of correlation between NGF-induced down-regulation of EGF receptors and NGF-induced neurite outgrowth in different PC12 cell variants investigated: different agents, PACAP and staurosporine, induced neurite outgrowth but not down-regulation of EGF receptors, and in suspension where neurite outgrowth is not possible NGF treatment induces down-regulation of EGF receptors in the absence of neurite outgrowth.
Preliminary studies show a significant decrease in EGF receptor mRNA after treatment of cells with NGF for 5 days.2 Under comparable conditions, the stability of the mRNA is unchanged. These data indicate that at least part of the mechanism by which NGF down-regulates the EGF receptor is transcriptional, but further experiments are necessary to confirm and extend these observations.
In summary, our data are consistent with the signal transduction mechanism of p140trk receptors (4) and strongly suggest a series of NGF-induced, neurite outgrowth-independent, p140trk-Src-Ras phosphorylation events in the down-regulation of EGF receptors in PC12 cells.
We thank Dr. Simon Halegoua for dominant-negative Src cells, Dr. Geoffrey Cooper for dominant-negative Ras cells, Dr. Lloyd Greene for PC12nnr5 cells, and Dr. David Kaplan for 6.24 cells.