Brain-derived neurotrophic factor stimulates b ... - Wiley Online Library

Journal of Neurochemistry, 2001, 79, 278±285

Brain-derived neurotrophic factor stimulates b-amyloid gene promoter activity by a Ras-dependent/AP-1-independent mechanism in SH-SY5Y neuroblastoma cells Yolanda Ruiz-LeoÂn and Angel Pascual Instituto de Investigaciones BiomeÂdicas, Consejo Superior de Investigaciones CientõÂ®cas, Madrid, Spain

Abstract The b-amyloid peptide, the major component of Alzheimerassociated plaques, derives from a larger b-amyloid precursor protein (APP), that is expressed in both neural and non-neural cells. Overexpression of APP actively contributes to the development of senile plaques and is considered a risk factor for the disease. APP expression is regulated by a variety of cellular mediators, among them ligands of tyrosine kinase receptors. In this study, we present evidence that brainderived neurotrophic factor (BDNF) modulates, in a dose- and time-dependent fashion, APP promoter activity in SH-SY5Y neuroblastoma cells transiently expressing the receptor TrkB. The APP promoter contains two potential AP-1 sites, and we examined whether or not protein kinase C (PKC) and the AP-1 sites of the promoter mediate the BDNF-induced stimulation of APP. Stimulation of APP promoter activity by BDNF was

not affected by the PKC inhibitor bisindolylmaleimide, or by dominant negative mutants of the AP-1 components Fos and Jun, which, however, blocked the response to phorbol esters. These results suggest that activation of the APP promoter by BDNF is largely independent of PKC and AP-1. In contrast, activated Ras increased APP promoter activity in SH-SY5Y cells, and a dominant negative mutant of Ras abolished BDNF-mediated promoter stimulation. Taken together, our results suggest a mechanism that involves activation of the Ras/MAP kinase signaling pathway, and phosphorylation of as yet unidenti®ed effectors which in turn can activate response elements within the APP promoter. Keywords: b-amyloid precursor protein (APP), brain-derived neurotrophic factor, neuroblastoma cells, promoter, protein kinase C, Ras. J. Neurochem. (2001) 79, 278±285.

Alzheimer's disease is a neurodegenerative disorder of the human CNS that causes mental deterioration and progressive dementia. The disease is characterized by a massive loss of neurons accompanied by neuropathological lesions, including the deposition of ®brillar aggregates of b-amyloid protein in affected brain regions (Glenner and Wong 1984; Masters et al. 1985). The major component of the amyloid deposits is the b-amyloid protein, a hydrophobic 39±43residue amino acid peptide which derives from a set of alternatively spliced b-amyloid precursor proteins (APP) that are encoded by a single gene located in human chromosome 21 (Selkoe 1994). Although at physiological levels APP appears to elicit neurotrophic effects (Mattson et al. 1993; Smith-Swintosky et al. 1994; Furukawa et al. 1996), APP overexpression might cause neuronal degeneration, by a mechanism that very likely involves increased formation of b-amyloid. APP is expressed ubiquitously in mammalian tissues and its expression can be regulated by a variety of stimuli, including growth factors, and ligands of tyrosine kinase receptors. Nerve growth factor (NGF), as well as

epidermal growth factor (EGF) or basic-®broblast growth factor (bFGF), stimulate the activity of the APP promoter and increase APP mRNA in mammalian tissues and cultured cells (Mobley et al. 1988; Konig et al. 1990; Forloni et al. 1993; Ohyagi and Tabira 1993; Lahiri and Nall 1995; Cosgaya et al. 1996; Ringheim et al. 1997). In PC12 cells, a rat pheochromocytoma cell line, it has been found that NGF

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Received April 11, 2001; revised manuscript received July 4, 2001; accepted July 23, 2001. Address correspondence and reprint requests to Angel Pascual, Instituto de Investigaciones BiomeÂdicas (C.S.I.C.), Arturo Duperier, 4, 28029 Madrid, Spain. E-mail: [email protected] Abbreviations used: APP, b-amyloid precursor protein; BDNF, brainderived neurotrophic factor; bFGF, basic ®broblast growth factor; CAT, chloramphenicol acetyl transferase; CRE, cAMP response element; CREB, cAMP response element binding protein; DMEM, Dulbecco's modi®ed Eagle's medium; EGF, epidermal growth factor; NGF, nerve growth factor; PCR, polymerase chain reaction; PKC, protein kinase C; PLC, phospholipase C; SDS, sodium dodecyl sulfate; TPA, 12-Otetradecanoylphorbol-13-acetate.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 79, 278±285

BDNF stimulates APP activity in SH-SY5Y cells 279

also affects the splicing of APP mRNA isoforms (Smith et al. 1991; Fukuyama et al. 1993), as well as the catabolism and secretory processing of APP (Refolo et al. 1989; Rossner et al. 1998; Villa et al. 2001). Brain-derived neurotrophic factor, a member of the neurotrophin family, binds to the TrkB tyrosine kinase receptor. As reported previously (Segal and Greenberg 1996; Kaplan and Miller 1997), at least two different pathways are activated following binding of BDNF to its receptor in primary neurons, a Shc-dependent pathway which results in activation of the Ras/MAP kinase signaling cascade, and another that involves phosphorylation and stimulation of phospholipase C-gamma (PLCg), which in turn may result in activation of protein kinase C (PKC) (Zirrgiebel et al. 1995). Both pathways result in the activation of the transcription factor AP-1, a heterodimer of Fos and Jun oncoproteins. In this study we show that BDNF induces APP promoter activity in SH-SY5Y neuroblastoma cells transiently transfected with a TrkB expression vector. Brain-derived neurotrophic factor-induced stimulation is reduced progressively in successive deletions of the 5 0 regulatory region of the APP gene, and the reduction appears to be coincident with the loss of the AP-1 recognition sequences located into the APP promoter at positions 2 45/239 (5 0 -TGACTCA) and 2 351/2345 (5 0 -TGATTCA). However, expression of dominant negative mutants of Fos or Jun, which inhibit activation of the APP promoter by the phorbol ester 12-Otetradecanoylphorbol-13-acetate (TPA), was unable to block the response induced by BDNF. Therefore, the effect exerted by this neurotrophin on the APP promoter appears to be AP-1 independent. This hypothesis was further con®rmed, as BDNF stimulates the activity of an APP promoter construct containing a point mutation in the sequence of the proximal AP-1 response element. In addition, bisindolylmaleimide, an inhibitor of PKC that effectively blocks the promoter response to TPA, did not affect BDNF-induced stimulation, proving that PKC activation does not mediate the neurotrophin effect. In contrast, expression of activated Ras mimics the effect of BDNF on the APP promoter, and the expression of a dominant negative mutant of Ras effectively blocks BDNF-induced response, thus, suggesting a Ras-mediated mechanism. Materials and methods Cell cultures SH-SY5Y cells were cultured in RPMI medium containing 10% fetal bovine serum (Gibco Life Technologies Ltd., Paisley, UK). PC-pAB1 cells were cultured in Dulbecco's modi®ed Eagle's medium (DMEM)-HEPES supplemented with 5% fetal bovine serum and 5% donor horse serum. Polyclonal antibody 369A against the cytoplasmic domain of APP was a generous gift of Dr Samuel E. Gandy and the monoclonal antibody 22C11, which

recognizes the N-terminus of APP, was purchased from Boehringer Mannheim (Germany). BDNF and the inhibitor bisindolylmaleimide used in cell treatments were from PeproTech Ltd. (London, UK) and Tocris Cookson Inc. (Avonmouth, UK), respectively. Reporter plasmids and expression vectors The chloramphenicol acetyl transferase (CAT) reporter plasmid containing the 21099/1105 fragment of the human APP gene has been described previously (Belandia et al. 1998). Progressive 5 0 deletions to 2487, 2307 and 215 bp were prepared by polymerase chain reaction (PCR) from the original 21099/1105 bp fragment, kindly provided by Dr Lahiri's laboratory (Lahiri and Robakis 1991), and subcloned into the BamHI site of pBL-CAT8 (KleinHitpass et al. 1988), a plasmid which lacks the AP-1-binding site present in the pUC backbone. The cDNA encoding the TrkB receptor was inserted into the EcoRI site of the expression vector CMV5, which contains the SV40 early promoter. Expression vectors for oncogenic rasval12 and dominant negative rasAsn17 (Cales et al. 1988; Feig and Cooper 1988), as well as dominant negative mutants of c-fos and c-jun, have been described previously (Olive et al. 1997; Ahn et al. 1998). Point mutants, within the proximal AP-1 recognition site, of reporter plasmids containing the 21099/1105 or 2307/1105 fragments of APP promoter were obtained by PCR, using a sense oligonucleotide 5 0 -GCCGGATCAGCTGACTCGCCTGGCTCTGAG-3 0 which contains the desired nucleotide changes, and following the protocol established for the Stratagene's Quick Change site-directed mutagenesis system. DNA transfection The SH-SY5Y cells were transfected in DMEM containing 10% fetal calf serum. The 10% serum-containing RPMI growing culture medium was replaced by DMEM 4±6 h before transfection, and the cells were cotransfected by the calcium phosphate coprecipitation method with 5 mg of reporter plasmids, 2 mg of TrkB expression vector, and 5 mg of carrier DNA (high molecular mass calf thymus DNA). One hundred nanograms of a luciferase reference vector was simultaneously used as an internal control for transfection ef®ciency. In addition, when needed, 5 mg of expression vector for the constitutive active mutant of ras, or 10 mg of expression vectors for the dominant negative mutants of ras, fos or jun, were used. In all cases the total amount of DNA among different transfections was kept constant by addition of an empty non-coding expression vector. After 16 h of incubation in the presence of calcium phosphate, the medium was discarded and washed with 5 mL of phosphate-buffered saline. Fresh RPMI medium containing 0.5% serum was added and the cells were then incubated for an additional 48 h in the presence or absence of BDNF or TPA. CAT activity was determined by incubation of [14C]chloramphenicol with cell lysate protein. After autoradiography, the non-acetylated and acetylated [14C]chloramphenicol was quanti®ed and the data expressed as the percent of acetylated forms after each treatment. Each experiment was repeated at least 2±3 times with similar relative differences in regulated expression. 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% sodium dodecyl sulfate (SDS)] containing the protease inhibitors phenylmethylsulfonyl ¯uoride (1 mm) and leupeptin (10 mg/mL). Equal


280 Y. Ruiz-LeoÂn and A. Pascual

Fig. 1 BDNF stimulates APP promoter activity in SH-SY5Y cells transiently expressing TrkB. CAT activity was determined in SH-SY5Y cells transfected with the 21099 APP±CAT promoter construct. (a) Results obtained in cells cotransfected with 2 mg of a TrkB expression vector, or a control non-coding vector (pCMV5) and incubated in the presence or absence of 10 ng/mL BDNF for 48 h.

(b) CAT activity was determined in cotransfected cells after incubation with increasing concentrations (1±100 ng/mL) of BDNF for 48 h, or with 10 ng/mL of BDNF for different periods. Results are expressed as fold induction over the control values obtained in BDNF-untreated cells. Each data point represents the mean of duplicate cultures with variations , 5±10%.

amounts, 40 mg, of cell extracts were electrophoresed in a 8% SDS±polyacrylamide gel, transferred to an immobilon poly(vinylidine di¯uoride) membrane and the cellular APP detected with the rabbit polyclonal antibody 369A. 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.

were used. Thereafter, a dose of 10 ng/mL BDNF was used in the following experiments.

Results BDNF stimulates APP promoter activity in TrkB expressing SH-SY5Y cells Transient transfection assays were carried out to determine whether BDNF affects the transcriptional activity of the APP promoter in neuroblastoma cells. The results obtained are illustrated in Fig. 1. SH-SY5Y cells were transiently cotransfected with a chimeric plasmid containing the 21099 to 1105 bp fragment of the human APP gene linked to the CAT reporter gene, in the presence and absence of an expression vector for the neurotrophin receptor TrkB. As shown in Fig 1a, the neurotrophin was unable to stimulate CAT activity in cells transfected with a non-coding expression vector, whereas BDNF induced a strong increase in promoter activity in cells expressing TrkB, thus proving that the response to BDNF is mediated by this receptor. Figure 1b shows the results obtained in cells transfected with TrkB and incubated in the absence and presence of different concentrations (1±100 ng/mL) of BDNF for 48 h or with 10 ng/mL of BDNF for different periods. As seen in Fig. 1b, BDNF stimulates APP promoter activity in a dose- and time-dependent manner. The effect was clearly observed at 48 h with a dose of 10 ng/mL BDNF. At this time the response increases progressively with doses of BDNF from 1 to 10 ng/mL and it was not induced further when higher concentrations (50 and 100 ng/mL) of BDNF

Identi®cation of DNA regions mediating the regulation of APP transcriptional activity by BDNF To map the DNA sequences of the APP promoter involved in the BDNF-induced response, progressively deleted fragments (21099, 2487, 2307 and 215) of the promoter were linked to the upstream region of the CAT gene and cotransfected with TrkB into SH-SY5Y cells. Figure 2 shows basal activity and the response to BDNF of different fragments of the APP promoter. Basal promoter activity was maximal with the 2487/1102 bp fragment and decreased

Fig. 2 Analysis of DNA regions mediating the response to BDNF. SH-SY5Y cells were transiently cotransfected with an expression vector for the TrkB receptor and plasmids containing progressive deletions of the APP promoter. Schematic representation showing the size of the APP constructs and the position of the two AP-1 elements has been included at the left of the graph. Data are expressed relative to CAT activity obtained with the construct containing the 21099 to 1105 bp fragment and are the mean ^ SD of CAT activities obtained from three separate experiments performed with duplicates. Numbers indicating in each group the fold induction over the corresponding control value have been also included in the graph.



AP-1-lacking fragment would suggest an effect exerted by BDNF on proximal promoter elements or components of the basal transcriptional machinery.

Fig. 3 Role of Ras on APP promoter activity. Cells were incubated in the presence or the absence of 10 ng/mL BDNF for 48 h after cotransfection with the 21099 APP±CAT plasmid, the TrkB expression vector, and vectors for the constitutive active mutant rasVal12, the dominant negative mutant rasAsn17, or with an empty expression vector. Data illustrate CAT activities relative to the control value determined in untreated cells, and are expressed as mean ^ SD values.

progressively following deletion to nucleotides 2307 and 215, thus suggesting the presence of a weak silencer located between nucleotide positions 2487 and 21099. Absolute values of CAT activity showed a similar pattern, being maximal in cells transfected with the construct extending to nucleotide 2487. However, when expressed as fold induction, stimulation by BDNF was similar in cells transfected with plasmids extending to nucleotides 21099 and 2487, suggesting that these sequences do not contribute signi®cantly to the response of the APP promoter to the neurotrophin. The stimulation was reduced slightly in the 2307 fragment which contains only one AP-1 site, and signi®cantly in the shortest (215/1102) fragment which lacks the AP-1 sites. The progressive decrease of BDNFinduced activity would be compatible with a AP-1-mediated response, whereas the residual response observed in the

Fig. 4 Effects of the PKC inhibitor bisindolylmaleimide on the BDNF-induced response. Cells transfected with the 21099 APP± CAT plasmid and an expression vector for TrkB, were incubated with 10 ng/mL BDNF or with 100 nM TPA. CAT activity was measured after 48 h incubation in the presence or absence of 10 mM bisindolylmaleimide. Data are expressed relative to the activity obtained in control untreated cells.

Implication of the ras oncogene in the regulation of APP expression by BDNF The Ras/MAP kinase pathway is one of the key signaling cascades responsible for transmitting signals from growth factor receptors to the nucleus (Cobb et al. 1994; Johnson and Vaillancourt 1994; Hill and Treisman 1995). In order to analyze the effect of activated Ras on APP gene expression, SH-SY5Y cells were transiently cotransfected with a reporter plasmid containing the 21099 to 1105 bp fragment of the APP promoter, and expression vectors for the constitutive active (rasval12), or the dominant negative (Ha-rasAsn17) mutants of ras. As illustrated in Fig. 3, constitutive expression of Ras leads to a signi®cant increase in APP promoter activity, of similar intensity to that induced by BDNF. Incubation with BDNF caused a small insigni®cant increase in CAT activity in cells expressing activated Ras. In contrast, the expression of a dominant negative mutant of ras does not affect basal CAT activity, and effectively blocks the BDNF-induced response. Taken together, these results suggest that activation of Ras mediates the stimulation of APP promoter activity induced by the neurotrophin. Activation of PKC does not mediate the response to BDNF Binding of BDNF to its receptor TrkB leads to activation of PKC in primary neurons (Zirrgiebel et al. 1995). To investigate whether PKC mediates the effects induced by BDNF on the APP gene in SH-SY5Y cells, we analyzed the effect of bisindolylmaleimide, a speci®c inhibitor of PKC. Cells cotransfected with the 21099 fragment of the promoter and a vector expressing the receptor TrkB, were incubated with or without 10 ng/mL BDNF for 48 h in the presence or absence of the inhibitor. As a control, cells were also treated with 100 nm TPA, which is known to activate

Fig. 5 Expression of dominant negative mutants of c-fos and c-jun blocks the effect of TPA, but not the effect of BDNF on the APP promoter. CAT activity was determined after 48 h incubation with or without 10 ng/mL BDNF or 100 nM TPA in cells cotransfected with a promoter-containing reporter plasmid, an expression vector for TrkB, and dominant negative mutants for c-fos or c-jun.



Fig. 6 Mutation of the proximal AP-1 sequence does not affect the stimulation of APP promoter activity by BDNF. Cells transfected with CAT reporter plasmids containing wild-type and mutated fragments of APP promoter were incubated in the presence or absence of BDNF or TPA. After 48 h incubation, CAT activity was determined and data expressed relative to the activity obtained in untreated cells transfected with the wild-type 21099 to 1105 bp promotercontaining plasmid. Data are mean ^ SD of CAT activities obtained from two separate experiments performed with duplicates.

PKC and stimulate the activity of the APP promoter in glial cells (Trejo et al. 1994). Figure 4 illustrates the effect of 10 mm bisindolylmaleimide on the BDNF- and TPAinduced responses. Both BDNF and TPA elicit a similar promoter response. However, whereas bisindolylmaleimide effectively blocked the effects induced by the phorbol ester on the APP promoter, CAT activity was similarly induced by BDNF in cells incubated in the presence or absence of inhibitor. This result shows that BDNF increases APP promoter activity by a PKC-independent mechanism. BDNF-induced stimulation is not mediated by the AP-1 recognition sites of the APP promoter To examine a possible role of the AP-1 sites located in the 5 0 -¯anking region of the human APP gene in the response induced by BDNF, we analyzed the effects of the neurotrophin in the presence of dominant negative mutants of the Fos/Jun family. As a control we also studied the effect of these mutants on the TPA-induced response. Cells cotransfected with the promoter, TrkB, and vectors expressing the

dominant negative mutants were incubated in the presence or absence of 10 ng/mL BDNF (or 100 nm TPA) for 48 h. As shown in Fig. 5, whereas dominant negative mutants of Fos and Jun blocked the response to TPA, BDNF increased CAT activity with similar potency in the different groups analyzed. This suggests that, in contrast to the effect of TPA on the promoter, the BDNF response is mediated by an AP-1-independent mechanism. To further prove an AP-1 independent mechanism, we examined the ability of BDNF to transactivate a mutated APP promoter in which the proximal AP-1 site has been replaced by a non-functional sequence. Two different fragments of APP promoter, containing the mutation were used (21099/1102, 2307/1102), and once again the response to TPA was determined as a direct control. As shown in Fig. 6, both BDNF and TPA increase activity of constructs containing the native AP-1 sequence. However, the response of the mutant promoters to BDNF was unaltered, whereas the effect of TPA was abolished completely. These results show that the proximal AP-1 site is suf®cient to mediate the promoter response to TPA, and also unambiguously demonstrate that BDNF induces APP promoter activity by an AP-1-independent mechanism.

BDNF increases APP expression in TrkB-expressing cells To con®rm that BDNF is relevant to APP transcription we have also analyzed the effects of BDNF on the levels of APP protein in PC-pAB1 cells, a subclone of the rat pheochromocytoma PC12 cell line stably transfected with a TrkB expressing vector. As illustrated in Fig. 7, BDNF increases the levels of both the intracellular and secreted isoforms of APP (Fig. 7a). In contrast, BDNF did not affect APP levels in parental PC12 cells (not illustrated). In addition, incubation with BDNF signi®cantly increased promoter activity in PC-pAB1 cells (Fig. 7b), thus con®rming the relevance of BDNF/TrkB in the regulation of APP gene expression. Fig. 7 BDNF induces APP expression in TrkB-expressing cells. (a) PC-pAB1 cells were incubated in the presence or the absence of 50 ng/mL BDNF for the periods indicated, and intracellular and secreted APP isoforms analyzed by Western blot. The polyclonal antibody 369A and the monoclonal antibody 22C11 were used to identify the cell-associated and secreted APP, respectively. (b) CAT activity was determined in PC-pAB1 cells transiently transfected with the 21099 APP±CAT reporter plasmid, and treated for 48 h in the presence or absence of 50 ng/mL BDNF.



Discussion As a precursor of the b-amyloid peptide, APP plays a central role in the development of Alzheimer's disease. Moreover, overexpression of APP has been considered a risk factor for this pathology. The expression, as well as the metabolism, splicing and secretion of APP are regulated by ligands of the membrane tyrosine kinase receptors (Mobley et al. 1988; Konig et al. 1990; Ohyagi and Tabira 1993; Cosgaya et al. 1996). BDNF, as well as its receptor TrkB, a member of the Trk family of protein tyrosine kinases, are highly expressed in the adult hippocampus, one of the most affected tissues in Alzheimer's disease. Moreover, a signi®cant reduction in BDNF levels in the hippocampus and frontal cortex of patients with Alzheimer's disease, has been reported (Connor et al. 1997). In addition, BDNF and TrkB are induced in hippocampal neurons following the induction of long-term potentiation (Dragunow et al. 1997), and this also suggests a role for this neurotrophin in memory formation. According to these reports the neurodegeneration may be, at least in part, secondary to an alteration in BDNF and/or TrkB levels, and the use of BDNF as a therapeutic agent in Alzheimer's disease has been proposed. In this study, we analyzed the regulation of APP gene expression by BDNF in SH-SY5Y, a human-derived neuroblastoma cell line. Transient transfection studies have demonstrated that BDNF induces APP promoter activity in TrkB-expressing cells. In contrast, BDNF was unable to induce APP promoter activity in SH-SY5Y cells not expressing the TrkB receptor. In the rat-derived pheochromocytoma PC-pAB1 cell line, a subclone of PC12 cells stably transfected with a rat TrkB-expressing vector, BDNF increases both the intracellular and secreted APP levels, as well as APP promoter activity, thus con®rming the role of this neurotrophin and its receptor TrkB in the regulation of APP gene expression. Several mechanisms and signaling pathways could be involved in this BDNF-induced effect. Upon binding of BDNF, several tyrosine residues in the TrkB receptor are rapidly phosphorylated, and this phosphorylation leads to the activation of different signaling events. Among others, activation of Ras/MAP kinase and PLC-gamma/PKC pathways has been said to follow BDNF stimulation in TrkBexpressing cells (Williams et al. 1998). In SH-SY5Y cells, we found that expression of Ras stimulates APP promoter activity, whereas expression of a dominant negative Ras mutant blocks the effect induced by BDNF. These results suggest that activation of Ras is involved in the neurotrophin-induced response. Activation of PKC by BDNF has been reported in rat cerebellar granule neurons (Zirrgiebel et al. 1995), and activation of PKC by phorbol esters is a suf®cient signal for increased APP gene transcription in cultured astroglial cells (Trejo et al. 1994).

We have also found that TPA stimulates APP promoter activity in SH-SY5Y cells. However, our results rule out a PKC-mediated mechanism for APP promoter activation in SH-SY5Y cells, as the effect of BDNF was observed even in the presence of bisindolylmaleimide, an effective PKC inhibitor. These results are similar to others observed with bFGF in the human neuroblastoma SKNMC cells. In these cells, the downregulation of PKC failed to attenuate bFGF-induced APP transcription (Ringheim et al. 1997). In contrast, our results show that TPA-induced stimulation of the promoter was abolished in the presence of the inhibitor, thus con®rming that the phorbol ester induces APP promoter activity through a PKC-dependent mechanism. The APP promoter contains two AP-1 recognition sites, which have been shown to be functional and to mediate the effects of phorbol esters in cells of glial origin (Trejo et al. 1994). In particular, it has been described how a single copy of the distal AP-1 recognition site fused to a heterologous promoter is suf®cient to confer a response to phorbol esters. Binding of AP-1 factors to the proximal recognition site has been also described (Lukiw et al. 1994). Both the Ras/MAPK and PLCg pathways can activate the transcription factor AP-1. Stimulation of AP-1-dependent transcription activity in CNS by BDNF has been described previously (Gaiddon et al. 1996), and BDNF induces the expression of the c-fos and c-jun oncogenes in CNS neurons (Gaiddon et al. 1996; Courtney et al. 1997). Therefore, binding of AP-1 factors to their recognition sites in the APP promoter could mediate the effect of BDNF in TrkBexpressing SH-SY5Y cells. We have found that stimulation of APP promoter activity by BDNF is decreased slightly in these cells following deletion of a region (2487/2308) containing the distal AP-1 site, and drastically reduced after deletion of a fragment (2307 to 215) which contains the proximal AP-1 site, hence suggesting a role of AP-1 in the response. However, the effect induced by BDNF on the APP promoter in SH-SY5Y cells is not reversed by dominant negative mutants of the AP-1 components Fos and Jun, which reverse the effect of TPA, thus suggesting an AP-1independent mechanism. This was further con®rmed because the response to BDNF was equally unaffected by a point mutation in the sequence of the proximal AP-1 site, that effectively blocks the effect induced by TPA. Our results show that different mechanisms and promoter sequences mediate the effects of BDNF and TPA on the APP promoter. Whereas stimulation by TPA requires activation of PKC and is mediated by AP-1 sequences within the promoter, the effect of BDNF seems to be mediated by a PKC- and AP-1-independent mechanism. The effect induced by BDNF in SH-SY5Y cells requires activation of Ras, and is very likely is mediated by transcription factors, other than AP-1, which should be identi®ed. The APP promoter has the typical structure of a housekeeping gene, lacking the TATA and CCAAT elements, but containing 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; Pollwein 1993; Hoffman and Chernak 1995; Kovacs et al. 1995; Vostrov et al. 1995; Bourbonniere and Nalbantoglu 1996). In addition to the AP-1 recognition sites located at positions 245 and 2351, a number of response elements could mediate the effect induced by BDNF on the APP promoter in neuroblastoma cells. Among others, sequences that effectively contribute to sustain the basal activity of the APP promoter under standard culture conditions (Quitschke and Goldgaber 1992; Hoffman and Chernak 1994), as well as other elements still unidenti®ed, could be involved in the neurotrophin-induced response. In particular, the response could be mediated by a potential cAMP response element (CRE) located at positions 274 to 263 of the APP promoter. It has been described that BDNF causes phosphorylation of cAMP response element-binding protein (CREB) by a Ras-dependent mechanism (Finkbeiner et al. 1997; Pizzorusso et al. 2000), and stimulates CRE-dependent transcriptional activity in CNS neurons (Gaiddon et al. 1996). Therefore, although experiments should be performed to analyze whether activation of CREB mediate the effects of BDNF on APP promoter in SH-SY5Y cells, our results are compatible with a mechanism involving a Ras/MAPK-induced phosphorylation of CREB. 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. Yolanda Ruiz-LeoÂn is recipient of a fellowship from the Spanish `Ministerio de EducacioÂn y Cultura'. We thank Drs D. K. Lahiri and N. K. Robakis for providing the APP promoter, Dr S. Gandy for the polyclonal antibody 369 A, Dr C. Vinson for the Fos and Jun dominant negative vectors, and Dr Hatanaka for the TrkB-expressing PC-pAB1 cells.

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