Regulation of secretases by all-trans-retinoic acid Anna Koryakina, Jessica Aeberhard, Sabine Kiefer, Matthias Hamburger and Peter Ku ¨ enzi Institute of Pharmaceutical Biology, University of Basel, Switzerland The importance of vitamin A and its active metabolite retinoic acid (RA) for cellular growth, differentiation, and death, as well as for embryonic development and vision, is well documented [1]. Growing evidence points towards an additional role of retinoids in the mature brain, with effects on sleep [2], synaptic plastic- ity, learning, and memory [3]. Several publications have described a crucial role of retinoid signalling in neurodegenerative diseases, par- ticularly in Alzheimer’s disease (AD) [4,5]. Although fibril-destabilizing [6] and neuroprotective [7] features of retinoids against amyloid beta (Ab)-induced toxicity have been demonstrated, the underlying mechanisms remain unknown. According to the amyloid hypothesis [8], Ab accu- mulation is one of the most important steps in AD pathology, and results from impaired amyloid precur- sor protein (APP) processing. Therefore, some emerg- ing therapeutic approaches involve modulation of APP cleavage via b-secretase inhibition, or a-secretase acti- vation, by, for example, activation of protein kinase C (PKC) [9]. Links between retinoids and PKC were con- vincingly demonstrated [10], and even pointed to direct binding between PKCs and RA receptors and control of transcriptional activity [11]. However, besides activation of a-secretases, little is known about their effects on b-secretase and c-secre- tase. Moreover, tumour-promoting activity limits the Keywords Alzheimer’s disease; PDBu (phorbol- 12,13-dibutyrate); PKC (protein kinase C); retinoic acid; secretases Correspondence P. Ku ¨ enzi, Institute of Pharmaceutical Biology, University of Basel, Klingelbergstrasse, 50, 4056 Basel, Switzerland Fax: +41 61 267 14 74 Tel: +41 61 267 15 44 E-mail: peter.kueenzi@unibas.ch (Received 12 January 2009, revised 19 February 2009, accepted 4 March 2009) doi:10.1111/j.1742-4658.2009.06992.x One of the emerging approaches for the treatment of Alzheimer’s disease aims at reducing toxic levels of Ab-species through the modulation of secretases, namely by inducing a-secretase or inhibiting b-secretase and⁄ or c-secretase activities, or a combination of both. Although there is increas- ing evidence for the involvement of retinoids in Alzheimer’s disease, their significance in the regulation of Ab-peptide production remains unresolved. Our work concentrated on the regulation of all secretases mediated by all-trans-retinoic acid (ATRA), and supports the hypothesis that ATRA is capable of regulating them in an antiamyloidogenic sense at the levels of transcription, translation, and activation. Apart from increased a-secretase activity, we show a complex chain of regulatory events, resulting in impaired b-secretase trafficking and membrane localization upon protein kinase C (PKC) activation by ATRA. Furthermore, ATRA demonstrates substrate specificity for b-site amyloid precursor protein-cleaving enzyme (BACE) 1 over nonamyloidogenic BACE2 in b-secretase regulation, which probably promotes competition for amyloid precursor protein between ADAM17 and BACE1. Additionally, we report enhanced secretion of solu- ble amyloid precursor protein a after ATRA exposure, possibly due to PKC activation, as pretreatment with the PKC inhibitor Go ¨ 6976 abolished all these events. Abbreviations Ab, antibody; AD, Alzheimer’s disease; ADAM, a disintegrin and metalloprotease; APP, amyloid precursor protein; ATRA, all-trans-retinoic acid; Ab, amyloid beta; BACE, b-site amyloid precursor protein-cleaving enzyme; CTF, C-terminal fragment; DAG, diacylglycerol; ER, endoplasmic reticulum; FACS, fluorescence activated cell sorting; PDBu, phorbol-12,13-dibutyrate; PKC, protein kinase C; PS, presenilin; RA, retinoic acid; sAPPa, soluble amyloid precursor protein derived by a-cleavage; SE, standard error. FEBS Journal 276 (2009) 2645–2655 ª 2009 The Authors Journal compilation ª 2009 FEBS 2645 use of several potential PKC activators, such as phor- bol esters. Therefore, an assessment of the action of RA(s) in AD might be valuable for AD patients, as retinoids have a long history of clinical use [11,12]. To elucidate the regulation of secretases, we exam- ined the effect of all-trans-retinoic acid (ATRA) on a-secretase [a disintegrin and metalloprotease (ADAM) 9, ADAM10, and ADAM17], on b-secretase [b-site amyloid precursor protein cleaving-enzyme (BACE) 1 and BACE2], and on the components of the c-secretase complex [presenilin (PS) 1 and PS2]. Results ATRA treatment upregulated mRNA and protein levels of a-secretases We first looked for induced mRNA levels of ADAM9 and ADAM10 upon administration of ATRA by real- time PCR in the human neuroblastoma cell line IMR-32 [13]. Treatment with 5 lm ATRA for 2, 4, 6, 24 and 48 h increased the quantities of both ADAM9 and ADAM10 mRNAs, peaking at 174% and 205%, respec- tively, of the corresponding control levels (Fig. 1A). Enhanced ADAM9 and ADAM10 transcription cor- related with increased protein amounts upon treatment with 5 lm ATRA or 1 lm phorbol-12,13-dibutyrate (PDBu). ATRA treatment led to maximal protein levels of 127% and 168% for ADAM9 and ADAM10, respectively (Fig. 1B), whereas the expression of ADAM17 remained largely unchanged or within the range of experimental error in response to ATRA treatment (Fig. 1A,B). Additionally, the localization of ADAM9, ADAM10 and ADAM17 rapidly changed in response to ATRA, as shown by confocal immunofluorescence analysis: all a-secretases showed strong translocation to the cellular membrane (ADAM9 and ADAM10) or to perinuclear compartments (ADAM17) after treatment with 5 lm ATRA for the time periods indicated (Fig. 1C). ATRA activated PKCa and PKCbII, leading to increased APP cleavage To check whether ATRA induces PKC signalling, we treated IMR-32 cells with different amounts of ATRA (0, 1, 2, 5 and 10 lm) for the time periods indicated (0, 5, 10 and 15 min), and observed clear phosphorylation of PKC with 5 lm ATRA using a pan-phospho-PKC antibody (Fig. 2A). To determine whether 5 lm ATRA induced activation of classic PKCs for the indicated time periods, we examined the expression, location and phosphorylation of PKCa and PKCbII. Both PKCa and PKCbII showed increased phosphorylation (Fig. 2C), and translocated to the cell membrane after 10 min of ATRA exposure (Fig. 2B). To study the effect of PKC stimulation on APP cleavage, IMR-32 cells were treated with 5 lm ATRA (6 h) or 1 lm PDBu (1 h). Proteins from the collected media were then analysed for soluble APPa (sAPPa) by western blot, and elevated release was found after ATRA exposure, indicating increased a-secretase activ- ity (Fig. 2D). The appearance of the soluble APP frag- ment derived by a-cleavage (sAPPa) was partly abolished by the addition of 1 lm Go ¨ 6976, a known inhibitor of PKCs [14]. Similarly, application of 1 lm PDBu for 30 min or 1 h resulted in appearance of the sAPPa fragment, an effect that was completely abol- ished by pretreatment with Go ¨ 6976. As this effect might point towards the possibility that ATRA is capable of reducing Ab levels, we tried to detect changes in intracellular and extracellular lev- els of total Ab and the amyloidogenic fragments Ab40 and Ab42, utilizing several approaches, such as ELISA or immunoprecipitation. Whereas extracellular Ab could not be detected at all, changes in intracellular levels remained negligible (not shown). ATRA-induced signalling affects BACE1 but not BACE2 To study the effect of ATRA on BACE1 transcription, we treated IMR-32 cells for 2, 4, 6, 24 and 48 h with 5 lm ATRA and performed real-time PCR. BACE1 mRNA levels increased to a maximum of 168% of the control level after 4 h of ATRA treatment, and these levels persisted for up to 24 h (Fig. 3A). This was con- sistent with increased protein amounts, which reached 143% of control levels after 24 h (Fig. 3C), as shown by fluorescence activated cell sorting (FACS) analysis. Exposure to PDBu, however, only insignificantly increased the BACE1 protein quantity, to 106% (Fig. 3C). At the same time, neither ATRA nor PDBu led to any significant changes in the protein levels of BACE2, a homologue of BACE1, and the differences perceived remained within the range of experimental error (Fig. 3C). Moreover, localization of BACE2 remained unaffected upon ATRA treatment (Fig. 3B), whereas BACE1, which initially localized in the cytoplasm and cell membrane in untreated controls, showed a massively changed distribution 3 h post-treatment (Fig. 3D). As BACE1 is synthesized in the endoplasmic reticu- lum (ER), and increased BACE1 mRNA levels were observed in our experiments, we examined its distribu- tion by confocal immunofluorescence microscopy, Regulation of secretases by retinoic acid A. Koryakina et al. 2646 FEBS Journal 276 (2009) 2645–2655 ª 2009 The Authors Journal compilation ª 2009 FEBS looking particularly for BACE1 localization in the ER. BACE1 was initially localized in both membrane and ER, and colocalized to some extent with calnexin, a widely used ER marker protein. ATRA exposure for 3 h resulted in increased BACE1 colocalization with calnexin (Fig. 3D). – 3 IMR-32 6 ADAM9 ADAM10 ADAM17 24 ADAM10 ADAM9 ADAM10 ADAM9 * * * * * ** ** ADAM17 * * ADAM17 A B C 5 µM ATRA (h) Change in protein amounts (%) Change in mRNA amounts (%) * P < 0.05 ** P < 0.01 Fig. 1. ATRA activated a-secretases in IMR- 32 cells. (A) mRNA levels of ADAM9 and ADAM10 increased in response to ATRA treatment, as shown by real-time PCR, whereas changes in ADAM17 levels remained within the standard error (SE). (B) As assessed by FACS analysis, ADAM9 and ADAM10 protein levels were increased by both 5 l M ATRA or 1 lM PDBu, whereas that of ADAM17 remained largely unchanged. Error bars: mean ± SE. (C) Rep- resentative confocal fluorescence images of ADAM9 (first row), ADAM10 (second row) and ADAM17 (third row) translocation in response to 5 l M ATRA addition are shown. ADAM9 and ADAM10 demonstrated time- dependent translocation to the cellular membrane, whereas ADAM17 translocation to the cytoplasm was seen after 3 h of treatment, and continually increased over the 24 h of testing. A. Koryakina et al. Regulation of secretases by retinoic acid FEBS Journal 276 (2009) 2645–2655 ª 2009 The Authors Journal compilation ª 2009 FEBS 2647 However, further incubation of IMR-32 cells with 5 lm ATRA (6 h and longer) provoked translocation of BACE1 to the plasma membrane (Fig. 3D). Analogous treatment with 1 lm PDBu also resulted in BACE1 trans- location to the membrane; this effect was completely abolished by the PKC inhibitor Go ¨ 6976 (Fig. 3E). ATRA affected the activity, transcription and localization of PS1 As APP cleavage is sequential and modulated by ATRA at the a⁄ b-secretase level, we also investigated possible modulation of c-secretase-dependent cleavage. We focused on PS1 and PS2, which are homologous transmembrane proteins forming the functional core of the c-secretase [15]. RT-PCR experiments revealed slightly increased PS1 mRNA levels after treatment with 5 lm ATRA for 24 h (Fig. 4A). This was accompanied by the appearance of the full-length PS1 and its active C-terminal fragment (CTF) in protein samples from cells treated with ATRA or PDBu (Fig. 4B). This effect was efficiently blocked by addition of 1 lm Go ¨ 6976. PS2 levels remained AB C D Fig. 2. ATRA treatment activated PKCs and caused increased secretion of sAPPa in IMR-32 cells. (A) Phosphorylation of PKC was induced by application of various con- centrations of ATRA, and reached a maxi- mum upon treatment with 5 l M ATRA. (B) Exposure to 5 l M ATRA for 10 min induced translocation of PKCa and PKCbII to the cellular membrane, as shown by confocal microscopy. (C) PKCa and PKCbII were immunoprecipitated using specific antibod- ies, separated by SDS ⁄ PAGE, and subse- quently probed with PKCa, PKCbII and phospho-PKC antibodies. The protein levels of PKCa and PKCbII remained similar, but their phosphorylation levels increased upon ATRA treatment. Densitometric analysis is shown for phosphorylation of PKCa and PKCbII, based on basal expression of PKCa and PKCbII, respectively, from three inde- pendent experiments. (D) Treatment with 5 l M ATRA for 3, 6 and 24 h, or 1 lM PDBu for 0.5 and 1 h, induced sAPPa secretion into the cell culture media. This effect was partly abolished by addition of the classic PKC inhibitor Go ¨ 6976, as shown by immu- noblot with concentrated media. Represen- tative results obtained in at least three experiments based on cell counts are shown, as well as densitometric analysis of three independent experiments. Regulation of secretases by retinoic acid A. Koryakina et al. 2648 FEBS Journal 276 (2009) 2645–2655 ª 2009 The Authors Journal compilation ª 2009 FEBS unchanged upon ATRA treatment, and the active PS2 form, a CTF of 23 kDa, remained undetectable (not shown). Both PSs partly colocalized with calnexin in the ER and nucleus in control cells, as assessed by DNA counterstaining with DRAQ5 in confocal immunofluorescence microscopy. Whereas PS1 displayed a weak increase in nuclear distribution (Fig. 4C), PS2 localization remained unchanged after 6 and 24 h of ATRA treatment (not shown). ATRA-dependent regulation of secretases in other cell lines Additional experiments, performed in the murine neuroblastoma cell line N2a and the human embryonic kidney cell line HEK293, basically confirmed the results obtained with IMR-32 cells. We observed similar increases in PKCa and PKCbII phosphorylation (Fig. 5A,B), as well as translocation to the cellular membrane, upon ATRA treatment (Fig. S1). Co-localisation analysis D BACE 1 5 µ M ATRA (h) – 3 Calnexin Merged Pearson’s coefficient Overlap coefficient 0.252 0.374 0.644 0.671 1 µ M PDBu (h) 6 1 IMR-32 0.174 0.235 0.113 0.232 1 + 1µM Gö6976 pre-treatment 0.262 0.421 E Co-localisation analysis ** P < 0.01 * P < 0.05 + 5 µ M ATRA (4 h) – B BACE 2 Change in protein levels (%) C BACE 2 BACE 1 A BACE 1 Change in mRNA levels (%) * ** ** ** Fig. 3. ATRA-induced regulation of BACE1 was partly dependent on PKC activation. (A) BACE1 mRNA levels increased in IMR-32 cells in response to ATRA treatment for the times indicated, as shown by real-time PCR analysis. Results from four independent experiments are given. Error bars: mean ± SE. (C) FACS analysis showed increased BACE1 protein levels in IMR-32 cells after 24 h of ATRA treatment, whereas those of BACE2 remained within the range of experimental error. Results from at least three independent experiments are given. Error bars: mean ± SE. (B) BACE2 was localized in the outer membrane, and this remained unchanged upon exposure to 5 l M ATRA. Repre- sentative images are shown. (D, E) Localization of BACE1 in IMR-32 cells in response to 5 l M ATRA (D) or 1 lM PDBu (E) was assessed by confocal microscopy. Colocalization analysis between BACE1 and calnexin was performed using IMAGEJ software. Colocalized areas are shown in white, and Pearson’s and overlap coefficients are provided for each merged image. (D) ATRA treatment for 3 h affected the BACE1 cytoplasmic distribution and increased BACE1 colocalization with the ER marker calnexin in IMR-32 cells. Prolonged ATRA exposure (6 h and longer) resulted in BACE1 translocation towards the cellular membrane. The images shown are based on visibility and not protein amount. (E) PDBu treatment (1 l M) led to BACE1 translocation, similar to that induced by ATRA (first row), but this was abolished by cotreatment with 1 l M Go ¨ 6976 (second row). A. Koryakina et al. Regulation of secretases by retinoic acid FEBS Journal 276 (2009) 2645–2655 ª 2009 The Authors Journal compilation ª 2009 FEBS 2649 Levels of secreted sAPPa increased in response to 5 lm ATRA (Fig. S2E) and 1 lm PDBu (not shown) treatments. Cotreatment with the PKC inhibitor Go ¨ 6976 partially diminished sAPPa secretion into cell media to control levels (Fig. S2E). However, changes in intracellular and extracellular levels of total Ab, Ab40 and Ab42 remained irrelevant or below the limit of detection. Changes in mRNA and protein levels of ADAM9 and ADAM10 in N2a and HEK293 cells in response to ATRA treatment matched the observations seen in IMR-32 cells. ADAM9 and ADAM10 displayed increased mRNA and protein quantities in N2a and in HEK293 cells (Fig. S2). Additionally, translocation to the cellular membrane (ADAM9 and ADAM10) or to perinuclear compartments (ADAM17) upon treatment with 5 lm ATRA were observed (Fig. S3). Enhanced transcription of PS1 corresponded with increased protein expression in all cell lines (Fig. S4A,B). This was accompanied either by stable expression of full-length protein in N2a cells or by enhanced cleavage of PS1 in HEK293 cells (Fig. 5C). Addition of 1 lm Go ¨ 6976 abolished this effect (Fig. S4B). All cell lines displayed a weak increase in PS1 nuclear distribution (Fig. S4C,D). Increased mRNA levels (Figs S5 and S6A) and protein levels of BACE1 (Figs S5 and S6C) were accompanied by its impaired trafficking and late translocation to the cellular membrane due to ATRA (Figs S5 and S6D) and PDBu treatment (Figs S5 and S6E) in all cell lines. ATRA distinguished equally between BACE1 and BACE2, and influenced nei- ther the expression (Figs S5 and S6C) nor localization (Figs S5 and S6B) of BACE2 in any of the cell lines tested. Discussion One strategy in AD treatment is aimed at protecting neurons from the production of toxic Ab species [16]. Reduction of Ab 40 ⁄ 42 levels is mainly achieved by modulation of secretases, namely by the induction of a-secretase activity, by inhibition of b-secretases and ⁄ or c-secretases, or by a combination of both. This study provided evidence that ATRA regulates all secretases at the levels of transcription, expression, and activation. PKC activators upregulate a-secretases, eventually promoting the antiamyloidogenic pathway [17]. Pub- lished data on positive and⁄ or negative PKC modula- tion by ATRA are controversial, which may be explained by a biphasic effect of ATRA on PKC activity [18]. We observed increased phosphorylation of PKCa and PKCbII in response to 5 lm ATRA treatment in all examined cell lines. It is generally accepted that classic and novel PKCs become activated by diacylglycerol (DAG), triggering localization to the cellular membrane. Endogenous DAG levels differ in various cell lines, and determine the PKC activation profile. The classic model for PKC activation involves its phosphorylation and transloca- tion from the cytosol to the binding domain on 5 µM ATRA (h) – 6 24 PS1/FL PS1/CTF 5 µ M ATRA (h) – 24 GADPH PS1 50 kDa 25 kDa 1 µM PDBu (h) – 1 1 PS1/CTF 25 kDa 1 µM Gö6976 – – + PS1/FL 50 kDa 5 µM ATRA (h) – 6 6 PS1/CTF 25 kDa 1 µM Gö6976 – – + PS1/FL 50 kDa Co-localisation analysis Presenilin 1 5 µ M ATRA (24 h) – + DRAQ5™ Merged 0.171 0.227 A B C Pearson’s coefficient Fig. 4. Modulation of PS1 upon activation of PKC. (A) Increased mRNA levels of PS1 were observed upon PKC activation by ATRA treat- ment, as shown by RT-PCR analysis. (B) In IMR-32 cells, immunoblot analysis revealed increased levels of PS1 after exposure to 5 l M ATRA or 1 l M PDBu. Inhibition of PKC by cotreatment with 1 lM Go ¨ 6976 blocked this increase in IMR-32 cells. (C) Representative confocal fluo- rescence images of PS1 in IMR-32 cells showed slightly increased colocalization with the DNA counterstain DRAQ5. Colocalization analysis was performed using IMAGEJ software. Colocalized areas are highlighted in white, and Pearson’s and overlap coefficients are provided for each merged image. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Regulation of secretases by retinoic acid A. Koryakina et al. 2650 FEBS Journal 276 (2009) 2645–2655 ª 2009 The Authors Journal compilation ª 2009 FEBS cellular membranes, a translocation that we observed in all cell lines. This event correlated with positive modulation of a-secretases. In contrast to other findings, we observed increased mRNA and protein levels of ADAM9. Notably, recent findings suggest that ADAM9 acts as an important regulator upstream of ADAM10 by shedding and releasing its catalytically active ectodomain [19]. These findings are consistent with transcriptional and transla- tional upregulation of ADAM10, and the enhanced APP cleavage seen in our experiments. The observed translocation of ADAM9 and ADAM10 to the cyto- plasmic membrane further supports the idea of APP cleavage at the membrane by these secretases. Our experiments showed a strong correlation between PKC activation, translocation of ADAM17 into the perinuclear space and sAPPa secretion into the extracellular space. As PKCa and PKCd – both classic PKC isoforms – are located in the ER [20], we believe that other APP cleavage sites, at the Golgi and in the ER, are also impaired as a consequence of PKC activation. b-Secretase cleaves APP within endosomes during APP recycling from the plasma membrane, as well as in the Golgi and ER [21,22]. As release of active ADAM17 ultimately occurs in the same com- partments, namely the Golgi and ER [23], we deduce that ADAM17 is the main BACE1 competitor for intracellular APP cleavage. We further explored the effects of ATRA on BACE1 at the level of transcription, translation, and activity, and found ATRA-dependent upregulation of its mRNA and ⁄ or protein levels. Poor correlation between increased BACE1 transcription and b-secretase activity has been previously reported, leading to the ideas of control at the level of translation [24] or its localization by phosphorylation [25]. Interestingly, BACE1 increas- ingly colocalized with the ER marker calnexin upon ATRA treatment in our experiments. As pro-BACE1 is predominantly located within the ER [26], this suggests that addition of ATRA leads to BACE1 accumulation within the ER by obstructing its maturation. After long-term treatment, BACE1 was mainly detected at the cellular membrane. This localization might further impair BACE1-derived APP cleavage, which typically occurs intracellularly, owing to its requirement for an acidic pH. Moreover, we believe that membranous BACE1 mainly consists of the fully matured form, as transportation of BACE1 is initiated by phosphorylation on its cytoplasmic tail, which occurs exclusively after full maturation only [25]. BACE2, a structural homologue of BACE1, was not affected by ATRA, despite its sequence homology. BACE2 processes APP within the Ab domain between Phe19 and Phe20, close to the a-secretase site [27], and has distinct transcriptional regulation and function [28]. BACE2 localization at the cellular membrane remained unchanged in any of the cell lines tested, which is possibly of interest for the antiamyloidogenic 5 10 5 µM ATRA (min) IP PKCβII – 5 10 15 p-PKC PKCβII HEK 293 5 µM ATRA (min) – 5 10 15 IP PKCα p-PKC PKCα 5 µM ATRA (min) – 5 10 15 IP PKCα p-PKC PKCα 5 µM ATRA (min) IP PKCβII – 15 p-PKC PKC βII N2a HEK 293 5 µ M ATRA (h) – 6 24 PS1 CTF 5 µM ATRA (h) – 6 24 PS1 CTF N2a A B C Fig. 5. Cell line-specific differences in response to PKC activation in N2a and HEK293 cells. (A, B) Immunoprecipitation followed by immunoblot analysis showed similar PKCa and PKCbII protein lev- els upon ATRA treatment, and both proteins showed increased phosphorylation in N2a (A) and HEK293 (B) cells. (C) ATRA treat- ment slightly increased the expression of both full-length PS1 and its active CTF domain in N2a cells (first line), but enhanced the cleavage of full-length PS1 to its active CTF form in HEK293 cells (second line), as shown by immunoblot. A. Koryakina et al. Regulation of secretases by retinoic acid FEBS Journal 276 (2009) 2645–2655 ª 2009 The Authors Journal compilation ª 2009 FEBS 2651 processing of APP, as constitutive a-cleavage occurs at the membrane [22]. To investigate whether PKC activation affects further steps associated with c-secretase cleavage, we studied the effects of ATRA and PDBu on PS1 and PS2 participation in the formation of the macromolecular c-secretase complex [15]. We could not identify any changes in the level of expression of PS2, which was mainly detected as a full-length protein of 52 kDa at any of the time points tested. Moreover, wild-type PS2 was weakly expressed in all cell lines examined, and ATRA had no effect whatsoever. PS1, on the other hand, showed delicate ATRA- dependent modification, and displayed slightly enhanced nuclear localization, with the most pro- nounced effect being observed after 24 h. PS1 could be detected both as full-length protein and as active endo- proteolytic CTF, and expression of both forms increased after exposure to ATRA at 6 and 24 h. Interestingly, PDBu treatment had only minor effects on full-length protein levels, but led to the appearance of substantial amounts of the endoproteolytic frag- ment. This effect was abolished by cotreatment with the PKC inhibitor Go ¨ 6976. Intriguingly, Walter et al. [29] reported processed PS1 CTF as an in vivo sub- strate for PKC, which indicates that the physiological and ⁄ or pathological properties of the active PS1 form might be regulated by activated PKC. Overall, the human cell lines (IMR-32 and HEK293) displayed faster and stronger responses to PKC stimu- lation, and showed more stable phosphorylation, than N2a, a cell line of murine origin. This might depend on variations in endogenous DAG levels, determining the PKC activation profile. We observed no marked differences in either the incubation time required for PKC stimulation and secretase activation, in the tran- scription ⁄ translation ratio, or in translocation of secre- tases between tested cell lines. In conclusion, ATRA treatment specifically shifts secretase-dependent APP cleavage towards the antiam- yloidogenic, owing to activation of PKCa and PKCbII. Both subsequently affect various steps and players involved in APP processing. However, ATRA- induced alterations appear to be modest in nature, and further research is therefore needed to assess their physiological significance. Experimental procedures Cell culture and treatment The human neuroblastoma IMR-32 cell line was main- tained in DMEM ⁄ F12 (1 : 1) (Invitrogen, Basel, Switzer- land), and the murine neuroblastoma N2a and human embryonic kidney HEK293 cell lines were maintained in DMEM (Sigma-Aldrich, Buchs, Switzerland). Media were supplemented with 10% heat-inactivated fetal bovine serum (Amimed, Basel, Switzerland), 100 UÆmL )1 penicillin ⁄ strep- tomycin (Invitrogen), and 2 mml-glutamine (Invitrogen). All cell types were grown in a humified atmosphere con- taining 5% CO 2 . ATRA, Go ¨ 6976 and PDBu were dissolved in dimethyl- sulfoxide and directly added to the medium for the times indicated. Go ¨ 6976 was added 30 min prior to ATRA or PDBu treatment, unless indicated otherwise. During pro- longed treatment, medium was exchanged every 2 days. Preparation of protein extracts and media samples Cells were collected, washed with ice-cold NaCl ⁄ P i (pH 7.4), and lysed in a hypotonic buffer (10 mm Hepes, pH 7.9, 60 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 0.5% NP-40) containing protease inhibitors [1 mm phenylmethanesulfonyl fluoride, 1· Complete Protease Inhibitors (Roche Diagnos- tics, Rotkreuz, Switzerland)]. Cytoplasmic extracts were collected, and cleared by centrifugation at 16 100 g for 30 min. Protein concentrations of extracts were measured using Coomassie Protein Assay Reagent (Sigma-Aldrich). Media were collected, snap frozen and stored at )80 °C. Before use, the media were thawed overnight at 4 °C, and then applied to Ultrafree MC filters (cut-off 30 kDa) (Milli- pore Corporation, Bedford, MA, USA). The samples were concentrated to 200 lL by centrifugation at 2300 g for 30 min, and the protein concentrations were measured as described above. Western blotting Cell lysates were separated by SDS ⁄ PAGE and blotted onto nitrocellulose membranes using standard procedures. Membranes were blocked and incubated overnight at 4 °C with specific primary antibodies (Abs), diluted in blocking buffer: anti-PKCa, 1 : 1000; anti-PKCbII, 1 : 1000; anti- actin, 1 : 4000 (all Santa Cruz, CA, USA); anti-phospho- PKC, 1 : 1000; anti-PS1, 1 : 500; anti-PS2, 1 : 1000 (all Cell Signaling Technology, Beverly, MA, USA); and 6E10, 1 : 1000 (Signet Laboratories, Dedham, MA, USA). Specific bands were tagged using horseradish peroxidase- conjugated secondary Abs, and detected with the ECL Plus System (Amersham Pharmacia Biotech, Little Chalfont, UK). Immunoprecipitation Immunoprecipitation was performed according to standard procedures. Briefly, cells were grown in 75 cm 2 flasks to Regulation of secretases by retinoic acid A. Koryakina et al. 2652 FEBS Journal 276 (2009) 2645–2655 ª 2009 The Authors Journal compilation ª 2009 FEBS 80% confluency, starved overnight, and subsequently trea- ted with 5 lm ATRA for the times indicated. Cells were harvested, washed with ice-cold NaCl ⁄ P i , extracted in 100 lL of lysis buffer (20 mm Tris ⁄ HCl, pH 7.4, 25 mm MgCl 2 , 0.05% NP-40, 1 mm dithiothreitol, 1· protease inhibitors), and cleared by centrifugation at 16 100 g for 2 min. Three hundred micrograms of total protein was incu- bated with 1 lg of PKCa or PKCbII (both from Santa Cruz) antibody in 500 lL of lysis buffer for 90 min at 4 °C. Protein complexes were precipitated by adding 40 lLofa 50% slurry of protein G Sepharose beads (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for 90 min at 4 °C, washed four times with wash buffer (20 mm Tris ⁄ HCl, pH 7.4, 25 mm MgCl 2 , 0.05% NP-40, 1 mm dithiothreitol, 120 mm NaCl), and dissolved by boiling with 30 lL of 1.5· Laemmli buffer for 3 min at 95 °C. Samples were resolved by SDS ⁄ PAGE and transferred to nitrocellulose mem- branes. Filters were blocked, and analysed using antibodies against phospho-PKC, anti-PKCa and anti-PKCbII (all from Cell Signaling Technology). RNA extraction, real-time PCR, and sequencing Total RNA was extracted using TRIZOL (Invitrogen), according to the manufacturer’s instructions, and tran- scribed to cDNA by a reverse transcriptase reaction using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) was per- formed for ADAM9, ADAM10 and BACE1, using the ABI PRISM 7700 System (Applied Biosystems). b-Actin was used as an endogenous reference to normalize the quantification of target mRNAs. Reactions were performed in triplicate, and threshold cycle (C t ) values were normal- ized automatically by the software. Following reverse transcription, the cDNAs for b-actin, ADAM9, ADAM10 and BACE1 were amplified under these conditions: one cycle of 52 °C for 2 min, one cycle of 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min, and melting curve analysis at 60–95 °C. The following primers were used: human b-actin forward, 5¢-GGACTTCGAGCAAGAGATGG-3¢; human b-actin reverse, 5¢-AGCACTGTGTTGGCGTACAG-3¢; murine b-actin forward, 5¢-AGCCATGTACGTAGCCATCC-3¢; murine b-actin reverse, 5¢ -CTCTCAGCTGTGGTGGTG AA-3¢; human ADAM9 forward, 5¢-GAATGAATCACG ATGATGGGAG-3¢; human ADAM9 reverse, 5¢-CCAGC GTCCACCAACTTATTAC-3¢; murine ADAM9 forward, 5¢-CTTAACATCCCGAAGCCTGAC-3¢; murine ADAM9 reverse, 5¢-CTCACTGGTCTTCCCTCTGC-3¢; human ADAM10 forward, 5¢-TTCAGGAAGCTCTGGAGGA A-3¢; human ADAM10 reverse, 5¢-TCCTGGTGTGCAC TCTGTTC-3¢; murine ADAM10 forward, 5¢-AGCAACAT CTGGGGACAAAC-3¢; murine ADAM10 reverse, 5¢-TTG CACTGGTCACTGTAGCC-3¢; human ADAM17 forward, 5¢-CCGCTGTGTGCCCTATGT-3¢; human ADAM17 reverse, 5¢-CCAGGACAGACCCAA-3¢; human BACE1 for- ward, 5¢-AGGTTACCTTGGCGTGTGTCG-3¢ ; human BACE1 reverse, 5¢-GAGGCAATCTTTGCACCAAT-3¢; murine BACE1 forward, 5¢-CACCATCCTTCCTCAGCAA TAC-3¢; murine BACE1 reverse, 5¢-GTAACAAACGGACC TTCCACTG-3¢; human PS1 forward, 5¢-GTTACCTGCA CCGTTGTCCT-3¢; human PS1 reverse, 5¢-CTCATCTTGC TCCACCACCT-3¢; murine PS1 forward, 5¢-CTCGCCAT TTTCAAGAAAGC-3¢; murine PS1 reverse, 5¢-CAGT GCGGGTAAATCTCCAT-3¢. Nested PCR amplifications were carried out in individual 50 lL reactions in a Perkin Elmer Thermocycler Gene- Amp 9700 (Applied Biosystems). All amplicons were checked by sequencing (performed by Microsynth, Balgach, Switzerland). Immunofluorescence microscopy and data analysis IMR-32, N2a and HEK 293 cells were fixed in 4% formal- dehyde in NaCl ⁄ P i for a minimum of 15 min at 4 °C, per- meabilized using 0.2% Triton-X (prepared in NaCl ⁄ P i containing 10% heat-inactivated fetal bovine serum), and then incubated with primary Abs. The Abs were diluted in NaCl ⁄ P i containing 10% heat-inactivated fetal bovine serum as follows: anti-PKCa, 1 : 50; anti-PKCbII, 1 : 50 (both Santa Cruz, CA, USA); anti-PS1, 1 : 100; anti-PS2, 1 : 100 (both Cell Signaling Technology); anti-ADAM9, 1 : 50; anti-BACE2, 1 : 100 (both AbD Serotec, Du ¨ sseldorf, Germany); anti-ADAM10, 1 : 50; anti-ADAM17, 1 : 50 (both Chemicon Europe Ltd, Chandlers Ford, UK); anti- BACE1, 1 : 100 (Merck Chemicals Ltd, Beeston, UK; cat. no. 195111); and anti-calnexin, 1 : 100 (BD Biosciences, Basel, Switzerland). The immunogen in antibodies against BACE1 is a synthetic peptide (CLRQQHDDFADDISLLK) corresponding to amino acids 485–501 at the C-terminus of BACE1. Cells were then washed three times with NaCl ⁄ P i and incubated for 1 h with affinity-purified Alexa-Fluor 488 goat anti-[rabbit IgG (H + L)], Alexa-Fluor 488 goat anti- [mouse IgG (H + L)] or anti-rabbit Texas Red (all Invitro- gen, Molecular Probes, Basel, Switzerland; diluted 1 : 1500 in NaCl ⁄ P i ). Nuclei were stained with DRAQ5 (Alexis; diluted 1 : 3000 in NaCl ⁄ P i ), and visualized with a Leica TCS SP scanning confocal microscope. Identical exposure times were used across conditions. Series of optical sections were taken at 1 lm intervals in line average mode with a picture size of 512 · 512 pixels, using Leica confocal soft- ware, version 2.5 (Leica Microsystems, Heidelberg GmbH), and analysed with imagej 1.37t software (http://rsb.info. nih.gov/ij/; National Institutes of Health, Bethesda, MD, USA). A. Koryakina et al. Regulation of secretases by retinoic acid FEBS Journal 276 (2009) 2645–2655 ª 2009 The Authors Journal compilation ª 2009 FEBS 2653 For colocalization analysis, pictures were converted to eight-bit grey scale images at a 0 < 255 fluorescence inten- sity range, and the threshold for each channel was deter- mined by colocalization threshold plug-in. These automatically determined threshold values were used in the next step of colocalization analysis, performed with jacop plug-in [21], and Pearson’s correlation and overlap coeffi- cients are shown (for details, see http://rsbweb.nih.gov/ij/ plugins/track/jacop.html). Merged images with white areas displaying the colocalization between BACE1 and calnexin or PS1 and DRAQ5 (DNA counterstaining) were generated using imagej colocalization finder plug-in. FACS IMR-32, N2a and HEK293 cells were fixed in 2% parafor- maldehyde in NaCl⁄ P i for 10 min at 37 °C, permeabilized using 90% ice-cold methanol, and then incubated with pri- mary Abs overnight at 4 °C. The Abs were diluted in NaCl ⁄ P i containing 1% BSA as follows: anti-ADAM9, 1 : 50; anti-BACE2, 1 : 100 (both AbD Serotec); anti- ADAM10, 1 : 100; anti-ADAM17, 1 : 100 (both from Chemicon Europe Ltd, Chandlers Ford, UK); anti-BACE1, 1 : 100 (Merck Chemicals Ltd, Beeston, UK); anti-Ab40, 1 : 50; anti-Ab42, 1 : 50 (both The Genetics Company Inc., Schlieren, Switzerland); and 6E10 Abs, 1 : 100 (Signet Laboratories). Cells were washed twice with 1% BSA ⁄ NaCl ⁄ P i and incu- bated for 30 min with affinity-purified Alexa-Fluor 488 goat anti-[rabbit IgG (H + L)] or Alexa-Fluor 488 goat anti- [mouse IgG (H + L)], diluted 1 : 1500 in 1% BSA ⁄ NaCl ⁄ P i ) (Invitrogen-Molecular Probes), and analysed on a Dako CyAn ADP LX 7 using summit 4.3 software (DakoCytomation, Fort Collins, CO, USA). Statistical analyses Real-time PCR data were quantified by applying the DDCt model, according to the equation ratio = (E target ) DCt (target) ⁄ (E reference ) DCt (reference) ,whereDCt target =Ct control )Ct treatment , DCt reference =Ct control )Ct treatment , and E is the amplifica- tion efficiency of a particular pair of primers. The amplification efficiency of each primer pair was determined experimentally, as previously described [30]. Additionally, the Ct values were normalized within the logarithmic phase with the highest PCR amplification efficiency by abi prism 7000 software. For statistical analysis by unpaired t-test, we assumed that both treatment and con- trol groups have a Gaussian distribution of DCt values, as well as equal variances. FACS data were quantified as described in manual for summit V4.3 software. The original method was published by Overton [31]. 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Reduction of Ab 40 ⁄ 42 levels is mainly achieved by modulation of secretases, namely by the induction of a-secretase activity, by inhibition of b -secretases and. have a long history of clinical use [11,12]. To elucidate the regulation of secretases, we exam- ined the effect of all-trans-retinoic acid (ATRA) on a-secretase