Nerve growth factor modulates the expression and secretion of β-amyloid precursor protein through different mechanisms in PC12 cells

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 1077±1084 1077

Journal of Neurochemistry, 2001, 77, 1077±1084

Nerve growth factor modulates the expression and secretion

of b-amyloid precursor protein through different mechanisms

in PC12 cells

Ana Villa, Maria JesuÂs Latasa and Angel Pascual

Instituto de Investigaciones BiomeÂdicas, Consejo Superior de Investigaciones CientõÂ®cas, Madrid, Spain

Abstract

The b-amyloid protein, component of the senile plaques found

in Alzheimer brains is proteolytically derived from the

b-amyloid precursor protein (APP), a larger membrane-

associated protein that is expressed in both neural and non-

neural cells. Overexpression of APP might be one of the

mechanisms that more directly contributes to the development

of Alzheimer's disease. The APP gene expression is regulated

by a number of cellular mediators including nerve growth

factor (NGF) and other ligands of tyrosine kinase receptors.

We have previously described that NGF increases APP

mRNA levels in PC12 cells. However, the molecular mechan-

isms and the precise signalling pathways that mediate its

regulation are not yet well understood. In the present study we

present evidence that NGF, and to a lesser extent ®broblast

growth factor and epidermal growth factor, stimulate APP

promoter activity in PC12 cells. This induction is mediated

by DNA sequences located between the nucleotides 2 307

and 2 15, and involves activation of the Ras±MAP kinase

signalling pathway. In contrast, we have also found that NGF-

induced secretion of soluble fragments of APP into the culture

medium is mediated by a Ras independent mechanism.

Keywords: amyloid precursor protein, nerve growth factor,

PC12 cells, promoter, Ras, synthesis and secretion.

J. Neurochem. (2001) 77, 1077±1084.

One of the characteristic features of Alzheimer's disease is

the presence of senile plaques in which the b-amyloid

protein, a 40±42 amino-acid peptide, is the major com-

ponent. This 4-kDa peptide is derived by proteolytic

cleavage from a set of alternatively spliced b-amyloid

precursor proteins (APP), which are encoded by a single

gene located on chromosome 21 (Selkoe 1994). The APP

gene is ubiquitously expressed in mammalian tissues, and

gives rise to three major APP messenger RNAs that encode

for the isoforms APP695, APP751 and APP770. The precursor

protein APP plays a central role in Alzheimer's disease, and

signi®cant alterations of APP expression might contribute to

its development (Zhong et al. 1994). The appearance of an

Alzheimer-like pathology in Down's syndrome, probably

caused by the trisomy 21-associated duplication of the APP

gene (Neve et al. 1988), the degeneration of neurones

overexpressing APP (Yoshikawa et al. 1992) or the appear-

ance of b-amyloid-immunoreactive deposits in transgenic

mice carrying the human APP cDNA (Quon et al. 1991;

Games et al. 1995), strongly suggest a positive correlation

between the overexpression of APP and the formation of

amyloid deposits.

Overexpression of the APP gene might result in an

aberrant processing of the amyloid precursor, which leads to

a higher concentration of amyloidogenic fragments, and is

associated with the formation of b-amyloid deposits in the

brain (Fukuchi et al. 1992). The APP gene expression is

regulated in different cell types by a variety of stimuli,

including phorbol esters (Yoshikai et al. 1990; Trejo et al.

1994), thyroid hormones (Belandia et al. 1998; Latasa et al.

1998) or retinoic acid (Konig et al. 1990). In addition, and as

NGF is considered to be of bene®t in Alzheimer's and other

neurodegenerative diseases, it is of interest to analyse the

effects induced by NGF and other neurotrophins on APP.

Received October 30, 2000; revised manuscript received February 2,

2000; accepted February 14, 2001.

Address correspondence and reprint requests to Dr Angel Pascual,

Instituto de Investigaciones Biomedicas (CSIC), Arturo Duperier 4,

28029 Madrid, Spain. E-mail: [email protected]

Abbreviations used: APP, b-amyloid precursor protein; CAT,

chloramphenicol acetyl transferase; DMEM, Dulbecco's modi®ed

Eagle's medium; EGF, epidermal growth factor; FGF, ®broblast growth

factor; NGF, nerve growth factor; PKC, protein kinase C.

Nerve growth factor (NGF), the best characterized neuro-

trophic factor, has been found to increase APP mRNA in

SH-SY5Y human neuroblastoma cells (Konig et al. 1990),

in mouse brain primary cultures (Ohyagi and Tabira 1993)

and in developing hamster brain (Mobley et al. 1988). In

PC12 cells, a rat pheochromocytoma cell line that

constitutively expresses APP, NGF has been reported to

in¯uence transcript levels (Cosgaya et al. 1996), splicing of

APP mRNA isoforms (Smith et al. 1991; Fukuyama et al.

1993), localization (Fukuyama et al. 1993), catabolism and

secretory processing of APP (Refolo et al. 1989; Rossner

et al. 1998). Moreover, it has also been suggested that APP

might act to mediate the effects induced by NGF on neurite

outgrowth (Milward et al. 1992). However, so far the

precise mechanisms involved in this regulation remain

largely unclear.

The trophic effects of NGF are mediated by the speci®c

tyrosine kinase receptor TrkA, and also by the low-af®nity

neurotrophin receptor p75NTR (Barbacid 1994; Segal and

Greenberg 1996). Nerve growth factor binds to the receptor,

and activates various intracellular signalling pathways that

mediate the phosphorylation of speci®c transcription factors,

the activation of target genes and ®nally the biological

responses induced by the neurotrophin. Accumulating

evidence indicates that the binding of NGF to its receptors,

p75NTR and TrkA, mediates the responses of

the neurotrophin on APP mRNA expression, splicing, and

protein secretion in PC12 cells (Rossner et al. 1998). In

addition, TrkA receptor gene expression is decreased in

nucleus basalis (Higgins and Mufson 1989), and parietal

cortex (Hock et al. 1998) of patients with Alzheimer's

disease, suggesting that this receptor could play a role in the

neurodegenerative process associated with this pathology.

We have previously reported that in PC12 cells NGF, as

well as epidermal (EGF) and basic ®broblast (bFGF) growth

factors, increase APP mRNA levels by a mechanism that

very likely involves activation of Ras (Cosgaya et al. 1996),

and is probably mediated by speci®c sequences of the

APP promoter (Lahiri and Nall 1995; Lahiri et al. 1999).

However, the exact mechanisms, and the precise signalling

pathways that mediate these effects of NGF remain elusive.

In this work we have examined the role of the ras

oncogene in the response induced by NGF on APP in PC12

cells, and also in two different subclones of PC12 stably

transfected with a dominant negative mutant of the ras

oncogene (M-M17-26), or with an activated ras oncogene

under control of the MMTV promoter (UR61). We have

determined the promoter activity induced by NGF or

dexamethasone in those cell lines, and partially analysed

the sequences of DNA that mediate the response of the APP

promoter to NGF. We have also examined whether the

mitogen-activated protein kinase cascade is involved in this

response by using several dominant negative mutants, and

®nally analysed the speci®c pattern of intracellular and

secreted isoforms of APP induced by NGF in these cells.

Nerve growth factor induces the APP promoter activity, and,

according to our results, this activation is mediated by Ras

throughout the MAP kinase signalling pathway. Nerve

growth factor-induced activation of the promoter causes a

speci®c increase of the intracellular content of APP. In

contrast, the release of APP isoforms to the culture medium

appeared to be quite Ras-independent. On the other hand, the

response to NGF was signi®cantly reduced in a small fragment

(2 15/1102) of the promoter that, however, maintains a

residual response likely to involve direct effects of NGF on

different components of the basal transcriptional machinery.

Materials and methods

Cell cultures

PC12 cells were cultured in RPMI-1640 medium containing 10%

donor horse serum and 5% fetal calf serum (GIBCO, Life

Technologies Ltd, Paisley, UK) in collagen treated plates. The

subclones of PC12 cells, UR61 and M-M17-26 cells, were grown in

a similar medium containing 10% horse ± 5% fetal calf serum.

UR61 cells were derived from PC12 cells following transfection

with a plasmid containing the transformant mouse N-ras oncogene

under the transcriptional control of the dexamethasone-inducible

mouse mammary tumour virus promoter (Guerrero et al. 1988).

The PC12 subline M-M17-26 was obtained by Szeberenyi et al

(1990) after transfection with the dominant negative mutant Ha-ras

(Asn-17) gene transcribed from the promoter of the mouse

methallothionein-1 gene. This subclone constitutively express

high levels of mutant Ras (Val-12) protein, which could not be

further induced by zinc. In our culture conditions both subclones, as

well as parental PC12 cells, exhibit more than 95% viability as

determined by trypan blue exclusion. In addition, the proliferative

ability of M-M71-26 cells is not affected by constitutive expression

of RasAsn17 (Szeberenyi et al. 1990). In UR-61 cells, RasVal12

induces differentiation; however, differentiation is not accompanied

by a decrease in the protein content per culture. The same occurs in

parental PC12 cells treated with NGF, whereas proliferative ability

of M-M17-26 cells is not signi®cantly affected by the neurotrophin.

The PC12 subclones were incubated with the different factors at

the concentrations and for the times indicated in the ®gures. NGF

and dexamethasone were obtained from Sigma (St Louis, MO,

USA), and bFGF and EGF were obtained from PeproTech EC Ltd.

(London, UK).

Plasmids

The chloramphenicol acetyl transferase (CAT) reporter plasmid

containing the 2 1099/1105 fragment of the human APP gene has

been previously described (Belandia et al. 1998). Progressive

5 0 deletions to 2 487, 2 307 and 2 15 bp were prepared by

polymerase chain reaction from the original 2 1099/1105 bp

fragment, kindly provided by Dr Lahiri's labouratory (Lahiri and

Robakis 1991), and subcloned into the BamH1 site of pBL-CAT8, a

plasmid that lacks the AP-1 binding site present in the pUC

backbone. Expression vectors for the dominant negative mutants of

Ras, Raf and MAPK have been previously described. These vectors

contain the inhibitory Ha-rasAsn17 mutant (Feig and Cooper 1988),

1078 A. Villa et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 1077±1084

a dominant negative Raf (Raf-C4) (Bruder et al. 1992), or the

Chinese hamster p44MAPK mutated in the kinase domain

(Meloche et al. 1992).

DNA transfection and determination of CAT activity

The PC12 cells were transfected in Dulbecco's modi®ed Eagle's

medium (DMEM) containing 10% fetal calf serum. The RPMI

growing culture medium was replaced by DMEM 4±6 h before

transfection, and cells were transfected by the calcium phosphate

coprecipitation method with 1 mg of reporter plasmids and carrier

DNA. One hundred nanograms of a luciferase reference vector was

simultaneously used as an internal control of the transfection

ef®ciency. In cotransfection experiments, 1 mg of reporter plasmid

and 10 mg of the corresponding dominant negative expression

vector were used. After 16 h of incubation in the presence of

calcium phosphate, the medium was discarded and washed with

5 mL of phosphate-buffered saline. A new RPMI medium con-

taining 0.5% serum was added and the cells were then incubated for

an additional period of 48 h in the presence or absence of NGF

(50 ng/mL). Each treatment was performed in duplicate cultures

that normally showed less than 5±15% variation in CAT activity,

which was determined by incubation of [14C]-chloramphenicol

with the same amount of cell lysate protein. After autoradiography,

the non-acetylated and acetylated [14C]-chloramphenicol were

quanti®ed. Each experiment was repeated at least 2 or 3 times

with similar relative differences in regulated expression. All data

are expressed as the mean ^ standard deviation.

Western blot analysis

Cellular proteins were extracted by lysis with a buffer (150 mm

NaCl, 50 mm Tris pH 8, 2 mm EDTA, 1% Triton, 0.1% SDS)

containing the protease inhibitors PMSF (1 mm) and leupeptin

(10 mg/mL). The protein content of cells was determined by using

the BCA assay (Pierce, Illinois, USA), according to the manu-

facturer's instructions. Identical amounts, 40 mg, of cell extracts

were then electrophoresed in an 8% SDS-polyacrylamide gel and

transferred to an immobilon polyvinylidine di¯uoride membrane.

Non-speci®c binding was blocked with 5% non-fat dried milk in

TBS-T (Tris buffered saline, 0.1% Tween 20) for 2±3 h at room

temperature and the cellular APP was detected with a 1/1500

dilution of the rabbit polyclonal antibody 369 A, raised against the

carboxi-terminal domain of human APP. After 1 h incubation at

room temperature (258C) the membrane was washed and incubated

with a second biotinylated anti-rabbit antibody (1/2000) for an

additional hour, washed again and ®nally incubated for 1 h with

1/2000 peroxidase-conjugated streptavidine. All incubations took

place at room temperature, and detection by enhanced chemilumi-

niscence (ECL, Amersham International plc., UK) was carried out

according to the manufacturer's indications.

Secreted full-length APP isoforms were detected by the same

method from 50 mL (1 : 100 from total) of conditioned medium

using the monoclonal antibody 22C11 at a ®nal concentration of

10 mg/mL. The apparent molecular mass (kDa) of the detected

bands was always determined by using a wide range protein

standard (Mark 12 from Novex, San Diego, CA, USA).

Results

Induction of APP promoter activity by NGF and other

neurotrophins

We have previously reported that NGF, as well as EGF or

bFGF, effectively increases APP-mRNA levels in PC12 cells

by a mechanism that requires activation of Ras (Cosgaya et al.

1996). To examine whether the speci®c changes induced by

these growth factors on the APP mRNA are induced at

transcriptional levels, we analysed the effect of NGF, EGF

and bFGF on the APP promoter activity. As shown in

Fig. 1(a), treatment with 50 ng/mLNGF for 48 h increased

by approximately four-fold the CAT activity in PC12 cells

transiently transfected with a chimeric plasmid containing

the APP promoter linked to the CAT reporter gene.

Epidermal growth factor and bFGF also stimulated APP

promoter activity, although their effect was weaker than that

Fig. 1 Regulation of APP promoter activity by NGF and other

neurotrophins in PC12 cells. Promoter activity was determined in

three different subclones of PC12 cells, the native cell line (a), the

M-M17-26 cell line that expresses a dominant negative ras mutant

(c), and UR61 cells which express the N-ras (val-12) oncogene in

response to dexamethasone (b). The cells were transfected with a

CAT reporter gene containing the 2 1099/1105 fragment of the APP

promoter, and CAT activity was measured after a 48 h period of

incubation in the presence or absence of 100 nM dexamethasone

(Dex), 50 ng/mL NGF, 17 ng/mL bFGF or 60 ng/mL EGF. CAT

activity was normalized for total protein, and results are expressed

as fold induction over the levels obtained in the corresponding con-

trol untreated cells. Data are the mean ^SD from three independent

experiments.

NGF regulates synthesis and secretion of APP by different mechanisms 1079


found with NGF. Because the ras oncogene is involved in

different actions of growth factor ligands of tyrosine kinase

receptors in PC12 cells, we examined the effect of activated

Ras on APP promoter activity in the PC12 subline UR61,

which contains a transfected N-ras oncogene (Rasval12)

under control of the glucocorticoid-inducible MMTV

promoter. Figure 1(b) shows that promoter activity was

signi®cantly induced in UR61 cells treated for 48 h with

100 nm dexamethasone. Because, as also shown in Fig. 1(a),

dexamethasone treatment of PC12 cells did not increase

promoter activity, these results indicate that activated ras is

responsible for promoter stimulation by the glucocorticoid

in UR61 cells. The induction of APP promoter activity by

dexamethasone in UR61 was comparable to that produced

by NGF in the parental PC12 cells. In contrast, NGF caused

a weaker increase in UR61 cells, in which the neurotrophin

does not elicit neurite outgrowth (Guerrero et al. 1988;

Cosgaya et al. 1997).

To determine whether endogenous Ras is required for

growth factor stimulation of the APP promoter in PC12

cells, we examined the ability of NGF, EGF and bFGF

to increase APP promoter activity in the PC12 subclone M-

M17-26, which as indicated above constitutively expresses

the dominant inhibitory RasAsn17 mutant. As illustrated in

Fig. 1(c), none of the three growth factors were able to

stimulate APP promoter activity in the presence of the

dominant negative ras mutant, showing the requirement of

functional Ras for this response.

Characterization of the signalling pathway that mediates

the effects of NGF on the APP promoter

The results obtained in M-M17-26 cells transfected in a

stable manner with the dominant inhibitory ras mutant were

con®rmed in parental PC12 cells transiently transfected with

RasAsn17. Figure 2 shows that transfection with this mutant

totally abolished the response to NGF in PC12 cells. As Raf

can act downstream of Ras in growth factor signal trans-

duction, the in¯uence of transient transfection with an

expression vector for a dominant negative form of Raf was

also analysed. As shown in Fig. 2, the mutant Raf had an

effect identical to that caused by RasAsn17, blocking the

response of the APP promoter to the neurotrophin in PC12

cells. The same was found with a MAP kinase mutant,

which also very signi®cantly reduced the promoter response

to NGF. In addition, transfection of these mutants decreased

basal CAT levels (data not shown), thus suggesting that

even in the absence of NGF there exists a certain activation

of the Ras/MAPK pathway that contributes to basal APP

promoter activity.

Identi®cation of DNA regions mediating the regulation of

APP transcriptional activity

To map the DNA sequences of the APP gene that mediate

the NGF-induced response, progressively deleted fragments

of the promoter (2 1099, 2 487, 2 307 and 2 15) were

linked to the CAT reporter gene and transfected into PC12

cells. As illustrated in Fig. 3, not only the basal activity, but

also the response to NGF was affected along the different

fragments of promoter studied. Deletion from nucleotides

2 1099 to 2 487 increased basal promoter activity without

affecting signi®cantly the response to NGF. Deletion to

nucleotide 2 307 caused a decrease of CAT levels similar to

those found with the fragment extending to nucleotide

Fig. 2 Stimulation of APP promoter activity by NGF is mediated by

the MAP kinase signalling pathway in PC12 cells. The CAT plasmid

containing the 2 1099/1105 fragment of the APP promoter was

transfected alone or in combination with dominant negative forms

(dn) of Ras, Raf and MAPK. CAT activity was determined in

untreated cells and in cells treated with 50 ng/mL NGF for 48 h.

CAT activity is expressed in each case as fold induction above the

control values found in NGF-untreated cells, and represent mean

^SD values of four determinations (duplicates from two independent

experiments).

Fig. 3 Analysis of DNA regions that mediate the effect of NGF on

the APP promoter. PC12 cells were transiently transfected with

pBL-CAT plasmids containing progressive deletions of the APP pro-

moter and CAT activity was determined in cell extracts after a 48 h

incubation in the absence or presence of NGF. Data are expressed

relative to CAT activity obtained with the construct containing the

2 1099/1 105 bp fragment and are the mean ^SD from two sepa-

rate experiments performed with duplicates.



2 1099. In addition, induction with NGF was similar,

indicating that sequences between nucleotides 2 307 and

2 1099 do not signi®cantly contribute to the response of

the APP promoter to NGF. However, further deletion of

sequences between 2 307 and 2 15 caused not only a strong

decrease of basal promoter activity, but also a marked reduc-

tion of the response to NGF, as only a residual response to

the neurotrophin was found in PC12 cells transfected with

the plasmid containing the fragment from 15 to 102.

Effects of NGF on the cell-associated proteins and the

accumulation of secreted forms into the culture medium

After 48 h of incubation in the presence or in the absence of

NGF (PC12 and M-M17-26 cells), or dexamethasone (UR61

cells), the content of APP was analysed in cells and culture

medium by western blot using the antibodies 369 A and

22C11, respectively. The analysis of the intracellular APP

isoforms is illustrated in Fig. 4(a). Based on previous

descriptions, the immunoreactive bands detected by western

blot analysis in PC12 cells contain at least six protein

species corresponding to immature and mature forms of

APP (APP695, APP751 and APP770; Buxbaum et al. 1990;

Weidemann et al. 1989). Incubation of PC12 cells with NGF

(50 ng/mL) for 48 h, does not affect the general pattern of

intracellular proteins (data not shown), but leads to a

generalized increase of the intracellular APP content, that is

not observed in the M-M17-26 cell line, which constitu-

tively expresses the dominant negative Ras mutant. In

contrast, incubation of UR61 cells with 100 nm dexametha-

sone, which induces the expression of the stably transfected

ras oncogene, leads to a similar increase in the intracellular

content of APP. In addition, dexamethasone did not affect

the intracellular content of APP in the native PC12 cells or

in M-M17-26 cells (data not shown). Figure 4(b) shows the

effects of NGF and dexamethasone on the levels of APP

soluble isoforms accumulated into the culture medium. As

expected, NGF treatment leads to an increased sAPP content

in the PC12 conditioned medium. However, and in contrast

to that observed with the intracellular APP content, the

amount of secreted isoforms was not modi®ed by dexa-

methasone in UR61 cells. Furthermore, the amount of sAPP

accumulated into the culture medium was increased in the

NGF-treated M-M17-26 cells.

Discussion

It has been extensively documented that NGF can modulate

the postranscriptional processing and secretion of APP in

PC12 cells (Refolo et al. 1989; Rossner et al. 1998; Rossner

et al. 1999). In addition, we have previously demonstrated

that NGF, as well as the growth factors bFGF and EGF,

increases APP-mRNA levels through a Ras-dependent

mechanism (Cosgaya et al. 1996).

In this study we have examined the effects of NGF on

APP expression in three variants of PC12 cells, the parental

cell line and two different subclones, which represent a good

model to investigate the role of Ras in NGF-induced effects

in PC12 cells. UR61 cells express oncogenic N-ras and

M-M17-26 cells contain a dominant inhibitory mutant of

ras. Transient transfection studies demonstrate that NGF and

to a lesser extent bFGF and EGF stimulate APP promoter

activity in PC12 cells. It has been reported that NGF can

stimulate APP promoter activity when these cells are treated

with the growth factor for a duration of 4 days prior to, and 4

days after transfection with a plasmid containing the APP

promoter (Lahiri and Nall 1995). We demonstrate here that

48 h of incubation with NGF after transfection are suf®cient

to activate the APP promoter. These results demonstrate that

NGF increases APP gene expression at transcriptional level.

Activation of the protein tyrosine kinase receptors

initiates a cascade of intracellular signalling events leading

to regulation of speci®c genes and ®nally to regulation of a

wide variety of cellular responses. Numerous proteins and

several second messengers, among them the Ras-MAP

kinase cascade (Muroya et al. 1992; Thomas et al. 1992;

Wood et al. 1992), have been implicated in the signalling

pathway that follows binding of neurotrophins to their Trk

receptors. Evidence that activation of endogenous Ras

mediates the effects of NGF on APP promoter activity

comes from the experiments with the PC12 subclone

M-M17-26, which constitutively expresses the dominant

inhibitory mutant of ras Asn-17. This mutant has a reduced

Fig. 4 Western blot analysis of cellular and secreted APP isoforms.

After 48 h of incubation in the absence or presence of NGF (PC12

and M-M17-26 cells), or dexamethasone (UR61 cells), the intracellu-

lar and secreted isoforms of APP were analysed by western blot

with the antibodies 369 A (a) and 22C11 (b), respectively. The ®gure

includes blots obtained in a representative experiment. The apparent

size (kDa) of the protein bands is indicated at the right by arrows.

The relative changes induced by the different treatments are indi-

cated by a number, at the bottom, representing the mean ^SD

obtained from three independent experiments.



af®nity for GTP and inhibition of endogenous Ras function

by this mutant has been suggested to occur through

competition with normal Ras for regulatory proteins that

promote nucleotide exchange (Feig and Cooper 1988).

Szerebenyi et al. (1990), have shown that the mutant blocks

the neurite outgrowth induced by NGF or FGF, as well as

the induction of different genes. Our present results

demonstrate that expression of the Asn-17 mutant also

blocks the stimulation of APP promoter activity induced by

NGF in PC12 cells, thus showing the requirement of

functional Ras for this response. The results obtained in

dexamethasone-induced UR61 cells support this hypothesis

further. In this subclone of PC12 cells the transfected ras

proto-oncogene is under control of a dexamethasone inducible

MMTV promoter. As dexamethasone does not affect the

expression of APP in PC12 cells, it can be assumed that Ras

is the signal transduction pathway used by dexamethasone to

increase the intracellular content of APP in UR61 cells.

Moreover, the results obtained with dominant negative

mutants of Ras, Raf and MAP kinase, strongly suggest that

NGF-induced stimulation of the APP promoter activity not

only requires activation of Ras, but also the complete

activation of the Ras-MAP kinase signalling pathway.

Many transcription factors that constitute ®nal targets of

speci®c transduction pathways (Hunter and Karin 1992)

bind to speci®c response elements in the regulated genes,

and mediate the effects induced by neurotrophins. The APP

promoter has the typical structure of a housekeeping gene,

lacking the TATA and CAAT elements. Its sequence is

extremely well conserved between rat, mouse and human

(Chernak 1993), and contains multiple positive and negative

regulatory elements and consensus sequences for the binding

of several transcription factors (Salbaum et al. 1988; La

Fauci et al. 1989; Vostrov and Quitschke 1997; Querfurth

et al. 1999; Wright et al. 1999). In agreement with previous

descriptions (Lahiri et al. 2000), we found that progressive

deletions of promoter sequences led to signi®cant variations

of the basal promoter activity. This activity was maximal in

the construct containing the 2 487 fragment of the APP

promoter, suggesting the presence of a silencer element

between this position and the nucleotide 2 1099, and it was

further decreased in successive deletions to nucleotides

2 307 and 2 15. Deletion to nucleotide 2 307 caused a

signi®cant decrease of basal activity, which could be

secondary to the loss of the `5 0-GC element/AP1 site'

tandem described in the 2 383/2358 region of the promoter

(Querfurth et al. 1999). Further deletion of sequences to

nucleotide 2 15 caused an additional reduction which

probably represents the loss of previously described

response elements that are essential to maintain the basal

promoter activity (Salbaum et al. 1988; La Fauci et al. 1989;

Vostrov et al. 1997; Querfurth et al. 1999).

The response to NGF was maintained in the plasmid that

contains the ®rst 307 bp of the 5 0 upstream sequence, but it

was signi®cantly reduced in the next deletion plasmid

extending to nucleotide 2 15. These results indicate that

stimulation of the APP promoter activity by NGF is mainly

mediated through sequences located between nucleotides

2 307 and 2 15. In addition the smallest promoter fragment

used (2 15/1102) retains a residual response that probably

involves direct effects of NGF on different components of

the basal transcriptional machinery, or alternatively the

existence of regulatory sequences located downstream of

the main transcriptional initiation site.

Our results strongly suggest that transcriptional activation

of APP gene expression is responsible for the NGF-induced

increase in APP transcripts previously described by us in

PC12 cells (Cosgaya et al. 1996). We have reported that

NGF increases APP mRNA levels in PC12 cells. Further-

more, expression of activated ras in UR61 cells also leads to

a signi®cant increase in APP transcripts, whereas the

expression of a dominant negative mutant of ras blocked

the induction of APP gene expression by NGF in the

M-M17-26 cell line. Stimulation of transcription should lead

also to an increase in the cellular content of APP proteins.

This was indeed observed in PC12 cells, where NGF treat-

ment resulted in a signi®cant increase in the intracellular

levels of the different APP isoforms. Moreover, our results

indicate that this effect was also largely dependent on Ras.

As also occurred with promoter activity (Cosgaya et al.

1996), the NGF-induced increase of cellular APP observed

in PC12 cells was not reproduced in the M-M17-26 subline,

which expresses the inhibitory mutant of Ras, and was

mimicked by dexamethasone-induced expression of Ras in

UR61 cells. According to our results NGF appears to induce

a generalized increase of the different APP species detected.

However, and since a complete de®nition of bands has not

been possible, we cannot exclude that, as previously described

(Fukuyama et al. 1993), NGF only affected the synthesis of

APP695, thus increasing speci®cally the levels of both the

immature and mature forms of APP695 in PC12 cells.

In addition, it has been also reported that NGF, as well as

other neurotrophins, regulates the secretion of sAPP in PC12

cells (Refolo et al. 1989; Fukuyama et al. 1993; Desdouits-

Magnen et al. 1998). In agreement with those descriptions

we have found that NGF increases the amount of sAPP

accumulated into the cultured medium in the parental PC12

cells. In contrast, expression of activated Ras in the UR61

subline was unable to increase the extracellular sAPP content.

In addition, the increased release of sAPP induced by NGF in

PC12 cells was not completely abolished in the M-M17-26

cells that express a dominant negative mutant of Ras. There-

fore, and contrarily to the effects induced by NGF on promoter

activity, levels of mRNA and cellular content of APP, the

regulation of APP secretion by NGF in PC12 cells appears

to be mainly mediated by a Ras-independent mechanism.

These results are in apparent contradiction with the descrip-

tion that activation of the MAP kinase cascade in PC12



results in the rapid secretion of sAPP isoforms. Never-

theless, it has been also reported that although activation of

MAP kinase promotes APP secretion, the inhibition of this

kinase is not suf®cient to reduce APP secretion (Rossner

et al. 1999). Taken together, these results are compatible

with the existence of a Ras-independent mechanism respon-

sible for APP secretion in response to NGF in PC12 cells. A

number of possible mechanisms could explain the speci®c

MAP kinase-dependent/Ras-independent regulation of APP

secretion by NGF. Other TrkA-interacting proteins, such as

PLC-g or PI 3-Kinase (Greene and Kaplan 1995), or

docking proteins such as FRS2, that are phosphorylated in

response to NGF stimulation (Kouhara et al. 1997; Hadari

et al. 1998) could be involved in the observed effect. In

addition, activation of protein kinase C (PKC) downstream

of PLC-g could contribute to Ras-independent activation of

MAP kinase through PKC phosphorylation of Raf (Sozeri

et al. 1992).

Our results demonstrate that NGF activates transcription

of the APP gene, thus increasing expression of APP. If this

also occurs in humans in vivo, the use of NGF, proposed as

a paliative treatment in Alzheimer's disease, could rather

result in a risk factor for the disease. However, the neuro-

trophins can also increase the release of neurotrophic sAPP

fragments, which very probably should be accompanied by a

reduced generation and release of the b-amyloid peptide. If

this is the case NGF could indeed be useful to de®ne new

therapeutic strategies for the disease. Therefore, it will be

of interest to analyse the effect of NGF on APP gene

expression and on the secretion of neurotrophic sAPP in

humans.

Acknowledgements

This research was funded by grants from the Spanish ComisioÂn

Interministerial de Ciencia y TecnologõÂa (SAF 97±0183) and

DireccioÂn General de InvestigacioÂn de la Comunidad de Madrid

(08.5/0036/1998. AV was the recipient of a fellowship from

Consejo Superior de Investigaciones CientõÂ®cas, Glaxo-Well-

come. We thank Drs G. M. Cooper and Dr A. Pellicer for the

PC12 subclones, D K Lahiri and N K Robakis for providing the

APP promoter and S. E. Gandy for the polyclonal antibody

369 A. We also thank Dr A. Aranda for critical reading of the

manuscript.

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Nerve growth factor modulates the expression and secretion of β-amyloid precursor protein through different mechanisms in PC12 cells