Activation of transcription of the human presenilin 1 gene by 12- O -tetradecanoylphorbol 13-acetate Martine Pastorcic 1 and Hriday K. Das 1,2 1 Department of Pharmacology & Neuroscience and 2 Department of Molecular Biology & Immunology, and Institute of Cancer Research University of North Texas Health Science Center at Fort Worth, Fort Worth, TX, USA We have recently identified an Ets element controlling over 90% of the basal expression of the human presenilin 1 (PS1) gene. We have also shown that Ets1 and Ets2 act as trans- activators of the PS1 gene by cotransfection experiments in SK-N-SH neuronal cells. The PS1 gene is widely but dif- ferentially expressed across tissues and the expression in brain appears to be restricted to neurons. To gain further insight into the regulation of the gene we have examined the regulation of PS1 by 12-O-tetradecanoylphorbol 13-acetate (TPA). SK-N-SH neuronal cells were treated with 0.2 l M TPA for 30 min to 24 h and the level of expression of the endogenous PS1 gene was measured by Northern blot ana- lysis. A two- to threefold increase in the level of PS1 mRNA appeared 4–8 h after the addition of TPA. A similar increase in transcription activity was observed in nuclear run off experiments, indicating that the increased mRNA level results from an activation in the initiation of transcription of PS1. Consistently, TPA also increased the level of PS1 pro- tein. No activation of the PS1 endogenous gene by TPA was observed in hepatoma HepG2 cells. Next we tested the effect of TPA on the expression of the PS1 promoter by trans- fecting fusion genes including various fragments of the PS1 promoter linked to a CAT reporter into SK-N-SH cells. TPA also stimulated the expression of the PS1CAT con- structs. Generally wild type constructs )687/+178, )118/ +178, )22/+178 including the short )35/+6 fragment showed a minor two- to threefold activation by TPA. Point mutations eliminating the )10 Ets motif or the )6 CREB/ AP1 motif did not decrease the stimulation by TPA. Thus TPA appears to activate the transcription of the PS1 gene by a mechanism which does not require these elements. Keywords: presenilin; transcription; TPA; SK-N-SH; PKC. Mutations in the presenilin 1 (PS1) gene are the cause of a majority of familial early onset Alzheimer’s disease (FAD) cases [1,2]. PS1 is an integral membrane protein involved in the regulation of gamma secretase cleavage generating amyloid beta protein [3] and appears to play a crucial role in the normal metabolism of beta amyloid precursor protein as well as in the pathological increase of the Ab42 cleavage product [4]. Furthermore, the global phenotype of PS1 knockout mice indicates that PS1 function is also required for mammalian embryogenesis, including CNS and skeletal development [5,6]. Hence the identification of the mecha- nisms controlling the expression of the PS1 gene should relate directly to understanding further the development and differentiation pathways and the pathogenesis of FAD. PS1 is differentially expressed in a variety of tissues [2] and brain expression is restricted to neurons [7–11]. We have previously identified the promoter sequences controlling the basal expression of the PS1 gene [12]. In particular we have identified at position )10 an Ets element which controls over 90% of the basal expression. Typically Ets factors act in conjunction with other transcription factors binding at adjacent sites [13,14]. A Ca 2+ /cAMP response element binding protein (CREB) as well as an AP1 consensus homology are located immediately downstream from the Ets motif. Recent data has shown that the )5CREB homology is required for activation of PS1 by N-methyl- D - aspartate (NMDA) in SK-N-SH cells [15]. TPA (12-O- tetradecanoylphorbol 13-acetate) is a known activator of protein kinase C- (PKC) and AP1-dependent transcription. Prolonged treatment by TPA induces morphological and functional differentiation in cultured neurons including SH-SY5Y human neuroblastoma cells and the parental cell line SK-N-SH [16–19]. We have examined the regulation of PS1 during short (< 24 h) exposure to 0.2 l M TPA in SK-N-SH cells. EXPERIMENTAL PROCEDURES Northern blot analysis SK-N-SH and HepG2 cells were grown to 75% confluency in MEM Eagle’s culture medium containing 12.5% (v/v) fetal bovine serum. The TPA treatment was started by replacing the culture medium with serum-free medium containing 0.2 l M TPA. After various incubation times (from 30 min to 48 h) cells were harvested and total RNA was prepared by guanidine thiocyanate extraction [20]. RNA samples (15 lg) were resolved on denaturing 1% Correspondence to H. K. Das, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107, USA. Fax: + 1 817 735 2091, Tel.: + 1 817 735 5448, E-mail: hdas@hsc.unt.edu Abbreviations: EMSA, Electrophoretic mobility shift assays; FAD, familial early onset Alzheimer’s disease; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; PKC, protein kinase C; PS1, presenilin 1; TPA, 12-O-tetradecanoylphorbol 13-acetate; wt, wild type. (Received 9 August 2002, revised 11 October 2002, accepted 22 October 2002) Eur. J. Biochem. 269, 5956–5962 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03320.x (w/v) agarose gels containing formaldehyde, blotted onto MSI nylon filters (Micron Separation Inc., Westboro, MA, USA), UV cross-linked and hybridized sequentially with DNA probes. Prehybridizations were for 2 h, and hybrid- izations were for 20 h, in 50% (v/v) formamide, 1 M NaCl, 10% (w/v) dextran sulfate, 1· Denhardt’s solution, 2% (w/ v) SDS and 0.1 mgÆmL )1 salmon sperm DNA at 42 °C. After hybridizations, filters were washed three times with 1· NaCl/Cit for 10 min at 24 °C and once for 10 min at 55 °C. The DNA probes used were labeled by random priming with [a- 32 P]dCTP to specific activity > 2 · 10 9 cpmÆlg )1 . The PS1 probe was the 1115 bp fragment from 429–1543 of the human presenilin 1 cDNA sequence clone cc44 (acces- sion number L76517) obtained by PCR amplification of the cDNA with the forward primer 5¢-GGAGCCTGCAAGT GACAACAGC-3¢ and the reverse primer 5¢-GCCATCAT CATTCTCTGCAACAG-3¢. The human glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) probe included the entire cDNA. Nuclear run off analysis of transcripts initiated during TPA treatment At the end of treatment with TPA, SK-N-SH cells were washed with NaCl/P i and harvested. Aliquots of 10 7 cells were resuspended into 1 mL of 10 m M Tris, pH 7.4, 10 m M NaCl, 3 m M MgCl 2 and 0.5% Igepal CA-630 (Sigma). The cells were allowed to lyze on ice for 5 min. Nuclei were then pelleted for 5 min at 500 g, washed once with same buffer and resuspended into 50 lLof50m M Tris, pH 8.3, 5 m M MgCl 2 ,0.1 m M EDTA and 40% (v/v) glycerol and stored at )70 °C. Transcription reactions were started by adding an equal volume of 10 m M Tris, pH 8, 5 m M MgCl 2 ,0.3 M KCl, 1 m M ATP, 1 m M CTP, 1 m M GTP and 5 m M dithiothreitol to the nuclei suspension with 10 lCi [a- 32 P]UTP. Mixtures were incubated for 30 min at 30 °C with agitation at 150 r.p.m. Reactions were stopped by adding 150 lL of buffer containing 0.5 M NaCl, 50 m M MgCl 2 ,2m M CaCl 2 ,10m M Tris, pH 7.4, and 40 lgÆmL )1 of RNase-free DNase I. DNase I treatment was stopped by adding 50 lLof5%(w/v)SDS,0.5 M Tris, pH 7.4, 0.125 M EDTA and 50 lg of proteinase K. After 30 min incubation at 42 °C, samples were extracted with phenol : chloro- form : isoamyl alcohol (25 : 24 : 1, v/v/v). RNA was pre- cipitated by adding 2 mL ice cold H 2 O containing 100 lg tRNA and 2.5 mL of 10% (v/v) trichloroacetic acid. After incubation on ice for 40 min the precipitates were collected by filtration onto 0.45 lm Milllipore HA filters. Filters were washed three times with 10 lL of 5% (v/v) TCA, 30 m M sodium pyrophosphate and transferred to vials containing 2mLof20m M Hepes, pH 7.5, 5 m M MgCl 2 ,1m M CaCl 2 and 20 lgÆmL )1 DNase I. After 30 min treatment at 37 °C reactions were stopped with 50 lLof0.5 M EDTA and 70 lL of 20% (w/v) SDS, and heat-treated at 65 °Cfor 10 min. Samples were then treated with proteinase K for 30 min at 37 °C and extracted with an equal volume of phenol. RNA was precipitated with 0.3 M sodium acetate. RNA pellets were resuspended in 1 mL 10 m M Tes, pH 7.4, 0.2% (w/v) SDS and 10 m M EDTA. An equal volume of the same buffer containing 0.6 M NaCl was added and nitrocellulose strips bearing DNA samples to be tested were added to the vials and incubated at 65 °C for 48 h. Membraneswerewashedwith2· NaCl/Cit, 1% (w/v) SDS at 24 °C for 30 min and at 65 °C for 15 min Filters were exposed for 24 h. DNA probes for presenilin 1, GAPDH and 18S RNA were the same DNA fragments used in Northern blotting. DNA was denatured in 50 lLof 0.1 M NaOH for 30 min at 24 °C. Solutions were neutral- ized by addition of 450 lL6· NaCl/Cit and applied to nitrocellulose membrane. Transfection assays SK-N-SH cells were transfected with PS1CAT fusion genes containing various fragments of PS1 sequences flanking the transcription initiation site [12]. Cells were seeded at a density of 10 4 Æcm )2 2 days before transfection. Transfection by calcium phosphate precipitation and glycerol shock were as described previously [12]. After glycerol shock cells were treatedwith0.2l M TPA or dimethylsulfoxide for 16–18 h in serum-free MEM. Promoter activity in different samples was compared using the amount of protein present in the cellular extracts as an internal control. Each experiment was repeated three times, with a minimum of triplicate tests of each construct and treatment. The ()118, +178) m6 PS1CAT construct contains a mutation within the )6 CREB motif from AATGACGA (wt) to AATcgaGA (m6). It was generated by PCR-based site-directed mutagenesis using the QuickChange kit from Stratagene and the complementary primers 5¢-CAGAGCCGGAAATCGAG ACAACGGTGAG-3¢ and 5¢-CTCACCGTTGTCTCGA TTTCCGGCTCTG-3¢ including the mutant CREB site with PS1CAT ()118, +178) as a template. Electrophoretic mobility shift assays Nuclear extracts from SK-N-SH cells treated with 0.2 l M TPA or dimethylsulfoxide for 5 h in serum–free MEM were prepared as described previously [12]. Electrophoretic mobility shift assays (EMSAs) included either a 32 P-labeled probe containing the wild type sequences () 22, + 6) or a mutation of the )10 Ets motif from GGAAA to ttAAA. Reactions were carried out by incubating 0.1–0.2 ng of probe with 2–5 lg of nuclear extracts in the presence of 1–2 lg of poly(dI-dC)Æpoly(dI-dC) in 10 m M Hepes, pH 7.9, 50 m M NaCl, 0.75 m M MgCl 2 ,0.1m M EDTA, 1m M dithiothreitol, 1% Igepal CA-630 (Sigma) and 10% (v/v) glycerol for 30 min at 4 °C. DNAÆprotein complexes formed were then analyzed by electrophoresis on nondena- turing 6% polyacrylamide gels containing 0.5% Igepal CA- 630. The electrophoresis buffer was 0.25 · TBE (89 m M Tris, 89 m M boric acid and 1 m M EDTA). The gels were prerun for 20 min, and sample electrophoresis was for 90 min at 10 V cm )1 at 4 °C. Western blotting SK-N-SH cells were washed twice with NaCl/P i and harvestedin2· sample buffer [0.1 M Tris/HCl, pH 6.8, 4% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol containing 200 lgÆmL )1 aprotinin, 100 lgÆmL )1 pepstatin, 50 lgÆmL )1 leupeptin and 10 m M benzamidine] [21]. The DNA was sheared with a 22-gauge needle and extracts were centrifuged at 14 000 g for 30 min at 4 °C. The supernatant was stored at )70 °C. Aliquots (25 lg) were fractionated by electrophoresis on 12% polyacrylamide Ó FEBS 2002 Regulation of the presenilin 1 gene (Eur. J. Biochem. 269) 5957 gels. Proteins were transferred to poly(vinylidene difluoride) (PVDF) membranes (Millipore). Membranes were blocked with 1% (w/v) BSA for 60 min at 24 °C, and incubated with a 1 : 1000 dilution of the primary antibody aPS1-N [21] in 1% (w/v) BSA for 60 min, and with 1 : 2000 dilution of the secondary antibody for 45 min. Blots were stained with ECL reagent (Amersham). The same blots were stripped in 60 m M Tris, pH 6.8, 2% (w/v) SDS, and 100 m M b-mercaptoethanol at 75 °C for 30 min and retested for the level of actin protein with 1 : 1000 aActin (sc-8432, Santa Cruz Biotechnology, CA, USA). RESULTS TPA treatment increases the level of PS1 mRNA in SK-N-SH cells SK-N-SH cells were treated with 0.2 l M TPA for increasing amounts of time from 30 min to 24 h. Total cellular RNA samples (15 lg) from each time point were analyzed by Northern blotting (Fig. 1A). The PS1 cDNA probe revealed a major transcript at about 3 kb (Fig. 1A) and a lesser amount of a larger mRNA of about 7 kb was also visible only in the samples with higher expression of PS1 (not shown). This is consistent with the size of 3 kb reported for PS1 mRNA and 7 kb for a minor transcript initiating at an alternative site [22]. No significant difference in mRNA level between control and TPA treated samples was observed before the 1 h time point as displayed in the histogram quantification of the Northern data (Fig. 1B). By 2 and 4 h TPA treatment increased PS1 mRNA level by twofold to threefold. Over longer treatment time (24 h) no significant difference was observed between TPA treated and control samples. The GAPDH mRNA level used as an internal control showed no difference over time or with TPA treatment. In the same experiment carried out with hepa- toma HepG2 cells the level of PS1 mRNA remained unchanged over time or in the presence of TPA (Fig. 1C, Table 1). Therefore treatment of SK-N-SH cells by 0.2 l M TPA results in a transient increase in the level of PS1 mRNA, showing a maximum at 4–8 h. TPA increases the rate of transcription initiation of the PS1 gene in SK-N-SH cells To determine whether the increase in the level of PS1 mRNA results from the activation of the transcription of the gene we have performed nuclear run-off assays (Fig. 2). We prepared nuclei from SK-N-SH cells treated with 0.2 l M TPA for 5 h. The transcripts already initiated within the nuclei at the time of harvest were allowed to elongate in vitro in the presence of 32 P-labeled ribonucleotides. The labeled RNAs were then purified and the level of specific mRNAs was quantified by hybridization to DNA probes for the human PS1 cDNA and 18S RNA immobilized onto nitrocellulose filters. The level of 18S transcription remained unchanged after TPA treatment and was used as an internal control to quantify the changes in PS1 transcription. The rate of PS1 transcription appeared to increase by 2.5– to threefold in the presence of TPA. Thus the increase in the level of PS1 mRNA observed by Northern blotting of total cellular RNA results from an increase in the rate of initiation of transcription of PS1. PS1 protein level increases with TPA To confirm and extend the previous observations we have examined the level of PS1 protein in SK-N-SH cells. Cellular proteins were fractionated by electrophoresis on 12% (w/v) polyacrylamide gels and analyzed by Western Fig. 1. TPA increases the level of PS1 mRNA in SK-N-SH cells. (A) SK-N-SH cells were incubated in the presence of 0.2 l M TPA (T) or dimethylsulfoxide (C) for increasing amounts of time from 30 min to 24 h as indicated above the lanes. RNA (15 lg) was fractionated on denaturing 1.4% (w/v) agarose gels and analyzed by Northern blot- ting. Membranes were sequentially hybridized with cDNA probes for the human PS1 and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) genes. (B) The level of transcription at each time point was quantified by laser scanning of the autoradiograms. The level of the PS1 3 kb transcript in each lane was expressed as its ratio to GAPDH mRNA in the same sample. The average level of the normalized PS1 mRNA at each time point was estimated with n ¼ 4orn ¼ 5ineach of three experiments. The histogram displays the ratio between the average level of PS1 mRNA in the TPA-treated samples and the average levelin dimethylsulfoxide controlat each time point. (C) HepG2 cells were incubated with TPA or dimethylsulfoxide and total RNA was analyzed by Northern blotting as described for SK-N-SH cells in (A). The average level of the normalized PS1 mRNA at each time point was estimated with n ¼ 3orn ¼ 4 in each of two experiments. In the 2 h control lane the PS1 band is partially masked by a gel artefact. 5958 M. Pastorcic and H. K. Das (Eur. J. Biochem. 269) Ó FEBS 2002 blotting using an antibody recognizing specifically the N terminus of the PS1 protein. Three species were detected: the full length PS1 appearing as a 45 kDa polypeptide, as well as a larger aggregated form and the 30 kDa N-terminal fragment (Fig. 3). After a 17-h TPA treatment the level of the full length 45 kDa species and aggregated form increased by 1.5– twofold. No significant increase in the 30 kDa N-terminal fragment protein was observed. Thus TPA treatment increases the level of the PS1 protein. The full length PS1 has a relatively short half-life, and it is normally cleaved by endoproteolysis into a 30 kDa N-terminal fragment and 17 kDa C-terminal fragment which are considerably more stable [23]. It is possible that any increase in newly synthesized PS1 in the presence of TPA does not appear against the background of the larger cellular pool of the stable 30 kDa form. Hence we observe an increase in the level of the PS1 protein by TPA treatment which is consistent with the increased mRNA level. DNA sequences required to confer activation of transcription of PS1 by TPA We have recently identified a promoter area required for efficient expression of the PS1 gene in SK-N-SH cells and HepG2 cells including DNA sequences from )35 to +178 flanking the transcription initiation site [12]. We have transfected SK-N-SH cells with PS1CAT fusion gene constructs containing various fragments of PS1 sequences from )687 to +178 inserted upstream from the CAT reporter gene. With constructs including sequences from Table 1. TPA does not alter the level of the PS1 mRNA in HepG2 cells. HepG2 cells were incubated with TPA or DMSO and total RNA was analyzed by Northern blotting as described for SK-N-SH cells in (A). The level of PS1 mRNA was quantified by laser scanning of the auto- radiograms and normalized with the level of GAPDH mRNA in the same samples. The average level of PS1 mRNA at each time point was estimated with n ¼ 3 or 4 in each of 2 experiments. 30¢ 1h 2h 4h 8h 24h Dimethylsulfoxide 3 ± 0.4 1.4 ± 0.4 2.7 ± 1 3.9 ± 0.8 1.06 ± 0.34 1.3 ± 0.02 TPA 2.5 ± 0.4 1.9 ± 0.6 2.6 ± 0.6 3.3 ± 0.8 0.97 ± 0.06 1.4 ± 0.3 Fig. 3. TPA increases the level of PS1 protein in SK-N-SH cells. SK-N- SH cells were treated with 0.2 l M TPA for 17 h and cell extracts were fractionated by electrophoresis on 12% (w/v) polyacrylamide gels and analyzed by Western blotting as described in Experimental procedures. Control extract (C) and TPA-treated extract (T) (25 lg) were loaded in lanes 1 and 2, respectively. The size of molecular mass markers is indicated in kDa alongside the gel. Arrows mark the position of the full length 45 kDa, the aggregated form and the 30 kDa N-terminal fragment. The same blot was stripped and the level of actin protein was analyzed as a control. Bands were quantified by laser scanning of the autoradiograms. The level of PS1 was normalized to actin and was determined in three distinct experiments. Values were analyzed by the paired t-test/ ANOVA method, and a value of P < 0.05 was considered significant. The average level of the aggregated form was 1.7 ± 0.36 (P < 0.05) in TPA-treated samples and 0.88 ± 0.15 in control sam- ples. The full length PS1 was 1.74 ± 0.2 in TPA samples and the control level was 0.94 ± 0.2 (P < 0.05). The 30 kDa species was 1.2 ± 0.28 in the TPA-treated samples and 0.98 ± 0.4 in the controls. All averages were derived from n ¼ 3. Fig. 2. Nuclear run-off analysis of the transcription of PS1 in the presence of TPA. SK-N-SH cells were incubated in the presence of 0.2 l M TPAfor5h.Nucleiwerethenpurifiedandusedintranscrip- tion run-off analysis to quantify the RNAs being actively transcribed at the time of harvest as described in Experimental procedures. Transcription was quantified by laser scanning of the autoradiograms. The changes in level of PS1 transcripts were quantified after normal- ization with 18S RNA. TPA increased transcription of PS1 by 2.8 (± 0.8) with n ¼ 3 in two independent experiments. Ó FEBS 2002 Regulation of the presenilin 1 gene (Eur. J. Biochem. 269) 5959 )687 to +178, )118 to +178, )22 to +178, )22 to +42 or the minimal promoter )35 to +6 the activation by TPA was two- to threefold (Fig. 4). Thus the minimal promoter )35 to +6 is sufficient to confer activation by TPA. This sequence interval contains an Ets element at )10 (Fig. 5) which is crucial for the expression of PS1. It also contains a sequence element sharing homology with the consensus CREB/AP1 binding motif immediately adjacent to the Ets site [24]. The effects of TPA on transcription are commonly mediated by AP1. Furthermore, Ets factors are known to act in conjunction with a number of other regulatory proteins including AP1. Thus we have tested the effect of a point mutation eliminating the AP1 homology (m6) as well as a point mutation abolishing the )10 Ets site (m1) (Fig. 5). M6 reduced the activity of the )118 to +178 construct by about twofold; however, the mutant promoter retained two- to threefold stimulation by TPA, similar to the )118 to +178 wild type construct (Fig. 4). Thus the )6 CREB/AP1 homology is not required for TPA activation. Similarly, the point mutation m1 eliminating the )10 Ets binding site did not abolish induction by TPA. This may indicate that neither the )10 Ets element, nor the )6CREB/AP1motif are required for stimulation by TPA. Changes in DNAÆprotein interactions over the )22/+6 region of the PS1 promoter in nuclear extracts from SK-N-SH cells treated with TPA We have used EMSAs to detect changes in the binding activity of the proteins recognizing specifically the )10 region of the PS1 promoter in nuclear extracts of SK-N-SH cells treated with TPA (Fig. 6). In dimethylsulfoxide-treated Fig. 4. DNA sequences required for activation of PS1 transcription by TPA. PS1CAT fusion genes containing various fragments of the PS1 promoter linked to the CAT reporter gene were transfected into SK-N- SH cells. The end-points of the promoter fragments used in each of the constructs are indicated below the graph. m1 is a mutation from CCGGAAATGACGA to CC ttAAATGACGA eliminating the )10 Ets site. In m6 the mutation to CCGGAAAT cgaGA eliminates the adjacent CREB and AP1 homologies (underlined) [24]. Fig. 5. PS1 promoter sequence. PS1 promoter sequence from )118 to +178. The endpoint of the 3¢ and 5¢deletions used in this study are indicated by arrows. The transcription initiation site is shown (+ 1). The position of the Ets, CREB and Sp1 binding sites are underlined. Fig. 6. Changes in DNAÆprotein interactions over the )22 to +6 region of the PS1 promoter induced by TPA treatment of SK-N-SH cells. (A) Nuclear extracts from SK-N-SH cells were prepared from cells treated with 0.2 l M TPA for 5 h as well as from cultures where the same dilution of dimethylsulfoxide was added (D). DNAÆprotein interactions over the ()22 to +6) region of the PS1 promoter were examined by EMSAs. The positions of the specific complexes are indicated. Extracts were preincubated with aEts1/2 (aElanes),an antibody recognizing specifically Ets1 and Ets2, for 45 min at 24 °Cin the absence of DNA probe. An antibody unrelated to Ets factors (anti- PS1 sc-1245, from Santa Cruz Biotechnology, CA, USA) was included in control lanes (C). The probe was added and incubation was con- tinued for another 20 min. Reactions were analyzed by electrophoresis on 6% (w/v) native polyacrylamide gels at 4 °C. Lanes 1–5 include the wild type probe, lanes 6–10 display binding to the probe containing a mutation (GGAA fi ttAA) within the )10 Ets motif. (B) Low exposure of the region of the gel including complex B. 5960 M. Pastorcic and H. K. Das (Eur. J. Biochem. 269) Ó FEBS 2002 nuclear extracts (D) the pattern of DNAÆprotein complexes observed with the PS1 probe produced the specific com- plexes A, B, C, D, E, F, G and H. These specific proteinÆDNA complexes (A–H) appear to be generated by proteinÆprotein interaction with Ets factors and other proteins [12]. These complexes (A–H) are found to be absent in assays with the Ets motif mutant probe similar to the data described previously [12]. TPA treatment appears to result in the loss of the specific complexes F and H, a decrease in complex B, as well as an increase in complexes A, C, D, E and G. Complexes A and G are eliminated by preincubation of the control or TPA treated nuclear extracts with anti-Ets1/2 Ig, indicating that at least these complexes involve interactions with Ets1/2. Therefore TPA treatment generally increased the specific interactions of the )10 region of the PS1 promoter with nuclear factors, including the amount of complexes involving Ets1/2 factors. DISCUSSION The loss of PKC is a prognostic element in the severity of neuronal damage resulting from ischemia in vivo [25]. The activation of PKC by TPA inhibits cell death in vitro through a complex set of pathways where different PKC isozymes appear to play opposite roles [26]. TPA increases the level of the expression of the endogenous PS1 gene in SK-N-SH cells at the level of initiation of transcription. TPA had no effect on the mRNA level in HepG2 cells (data not shown), thus the regulation pathway implicated here may be somewhat cell specific. Most of the known biological effects of TPA are attributed to its ability to activate PKC. The effect(s) of TPA observed here are likely to result from the activation of PKC because the increase in PS1 mRNA appears to be abolished by bisindolylmalei- mide, a specific inhibitor of PKC [27,28], in preliminary data (not shown). Furthermore, the time course of activation of PS1 indicates that the maximum increase in the level of PS1 mRNA is reached by 4 h and that a longer exposure to TPA (24 h) no longer activates PS1 expression. This is consistent with the down-regulation of protein kinase C with long exposure to TPA observed in many cell types [29]. In order to analyze further the mechanism of activation we have tested the effect of TPA on the activity of the PS1 promoter in transient infection assays in SK-N-SH cells. TPA treatment activated similarly by two- to 2.5-fold the transcriptional activity of all promoter fragments tested. The minimal promoter including sequences )35/+6 appears to retain TPA activation. Mutations eliminating the )10 Ets binding or the )6 AP1/CREB motifs did not reduce activation by TPA. This suggests that induction results from the modification of protein(s) of the initiation complex which do not bind directly to DNA. They may however, interact with factors recognizing specific motifs, such as Ets, and promote changes in proteinÆDNA interac- tions within complexes including Ets. For example there is a significant increase in the amount of the larger complexes A and B (Fig. 6) in the TPA treated extracts. Complex A is likely to contain Ets1 or Ets2, as it is eliminated by the addition of anti-Ets1/2 Ig. It is possibly converted into complex B (which increases from lane 4 to lane 5). This may indicate that Ets 1/2 is not required for the formation of complex B. The identity of the protein recognizing specif- ically the PS1 promoter within B is not known. However its ability to interact directly or indirectly with Ets1/2 should enable its identification by the 2-hybrid selection technique. Members of the AP1 protein complex have been impli- cated in the onset of apoptosis. Induction by c-fos is an early event in apoptosis [30], the overexpression of c-jun domin- ant negative mutants protects sympathetic neurons against programmed cell death induced by the withdrawal of nerve growth factor whereas overexpression of wild type c-jun appears to trigger apoptosis [31]. Retinoic acid-induced apoptosis in F9 cells also induces c-jun, and the reduction of c-jun levels by antisense reduces apoptosis [32]. In contrast, the same F9 cells stably transformed with wild type PS1 show a significantly reduced level of apoptosis after retinoic acid treatment, whereas mutant PS1 suppresses apoptosis only weakly. This indicates that PS1 may play a protective role in the development of c-jun-mediated apoptosis. Thus the induction of PS1 gene expression after treatment with TPA in the experiments described here is consistent with a role of PS1 in the c-jun cascade leading to apoptosis. However, the role of the c-jun N-terminal kinase (JNK)/ c-jun cascade for in vivo apoptosis and particularly in Alzheimer’s disease is still unclear [33]. Growing evidence implicates JNK-dependent pathways in Ab-dependent apoptosis [34]. A role of PS1 in the development of Ab-induced apoptosis has previously been suggested [35]. Overexpression of mutant PS1 increased the susceptibility of PC12 cells to apoptosis induced by Ab or the withdrawal of trophic factors. In contrast with this proapoptotic effect of PS1 mutants, the wild type PS1 suppresses apoptosis induced by the activation of p53 [36], which is a target of JNK. 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Activation of transcription of the human presenilin 1 gene by 12 - O -tetradecanoylphorbol 13 -acetate Martine Pastorcic 1 and Hriday K. Das 1, 2 1 Department. 10 9 cpmÆlg )1 . The PS1 probe was the 11 15 bp fragment from 429 15 43 of the human presenilin 1 cDNA sequence clone cc44 (acces- sion number L76 517 ) obtained by PCR