Báo cáo khoa học: 7-Ketocholesterol-induced apoptosis Involvement of several pro-apoptotic but also anti-apoptotic calcium-dependent transduction pathways ppt

7-Ketocholesterol-induced apoptosis Involvement of several pro-apoptotic but also anti-apoptotic calcium-dependent transduction pathways Arnaud Berthier, Ste ´ phanie Lemaire-Ewing, Ce ´ line Prunet, Thomas Montange, Anne Vejux, Jean Paul Pais de Barros, Serge Monier, Philippe Gambert, Ge ´ rard Lizard and Dominique Ne ´ el INSERM U498 – Me ´ tabolisme des lipoprote ´ ines et interactions vasculaires, Dijon Cedex, France Oxysterols are probably the components of oxidized low-density lipoproteins which have the strongest involvement in the genesis and development of athero- sclerosis [1–3]. Oxysterols mediate the early events of atherosclerosis observed during the development of the disease, such as the production of proinflammatory cytokines, expression of adhesion molecules, and cyto- toxicity to the cells of the vascular wall and mono- cytes ⁄ macrophages [4–6]. This cytotoxicity appears to be mainly related to the induction of apoptosis [6]. Among cytotoxic oxysterols found in atheromatous lesions, 7-ketocholesterol is one of the most abundant and one of the most studied [7]. In a previous report, we demonstrated the involvement of the calcium-dependent activation of calcineurin (PP2B) leading to dephosphorylation of the pro-apoptotic protein BAD in 7-ketocholesterol-induced apoptosis of THP-1 cells. The rise in free-Ca 2+ activating calcineu- rin is induced by the translocation of Trpc-1, a com- ponent of the store-operated Ca 2+ entry channel, into lipid raft domains, which are microdomains of the plasma membrane formed by the lateral packing of glycosphingolipids and cholesterol [8]. However, in this study, we show that BAD was dephosphorylated at serine 99 prior to dephosphorylation at serine 75, and the use of calcineurin inhibitors does not completely inhibit BAD dephosphorylation, suggesting the activa- tion of other pro- or anti-apoptotic pathways. Keywords apoptosis; calcium; 7-ketocholesterol; signal transduction; THP-1 cells Correspondence D. Ne ´ el, INSERM U498 – Laboratoire de Biochimie Me ´ dicale, CHU ⁄ Ho ˆ pital du Bocage, 2 Bd Mare ´ chal de Lattre de Tassigny, BP 77908, 21079 Dijon Cedex, France Fax: +33 3 80 29 36 61 Tel: +33 3 80 29 50 03 E-mail: dominique.neel@chu-dijon.fr (Received 28 January 2005, revised 11 April 2005, accepted 18 April 2005) doi:10.1111/j.1742-4658.2005.04723.x Oxysterols, and particularly 7-ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and mono- cytes ⁄ macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by a sustained increase of cytosolic-free Ca 2+ , which elicits the mitochondrial pathway of apoptosis by activation of the calcium-dependent phosphatase calcineurin, leading to dephosphorylation of the ‘BH3 only’ protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we dem- onstrate the involvement of two other calcium-dependent pathways during 7-ketocholesterol-induced apoptosis. The activation of the MEK fi ERK pathway by the calcium-dependent tyrosine kinase PYK 2, a survival path- way which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro-apoptotic BH3 only protein, Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells induces the release of Bim-LC8 from the microtubule-associated dynein motor complex, and its association with Bcl-2. Therefore, it appears that 7-ketocholesterol-induced apoptosis is a complex phenomenon resulting from calcium-dependent activation of several pro-apoptotic pathways and also one survival pathway. Abbreviations MEK 1 ⁄ 2, MAPK-Erk kinase-1 and )2; MSB, microtubule-stabilizing buffer; PTK, protein tyrosine kinases. FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3093 Of the various pathways that are involved in cell survival, the Ras ⁄ Raf ⁄ MEK ⁄ Erk pathway plays a crit- ical role. Indeed, Ras-activated Raf operates by phos- phorylating and activating MAPK-Erk kinase-1 and -2 (MEK 1 ⁄ 2) [9,10]. MEKs have very narrow sub- strate specificity, restricted to p44 Erk1 and p42 Erk2 . Phosphorylation of these kinases by MEKs results in phosphorylation of further downstream targets inclu- ding p90 Rsk , which could phosphorylate and inactivate BAD at serine 75 [11]. On the other hand, it has been shown that protein tyrosine kinases, such as proline- rich tyrosine kinase-2 (PYK 2), transduce key extracel- lular signals through the activation of the MEK ⁄ Erk pathway [12]. Moreover, studies have shown that PYK 2 is activated by an increase in intracellular Ca 2+ concentration, confirming the well-known pro- cess of Ca 2+ -induced ERK activation [13]. Bim, like BAD, is a ‘BH3-only’ protein of the Bcl-2 family and an important mediator of apoptosis in response to loss of survival signal [14,15]. There are three major splice variants of Bim: short (Bim S ), long (Bim L ) and extra-long (Bim EL ) [16]. In most cells, including THP-1 cells, Bim EL is the major species expressed [17]. This isoform of Bim induces apoptosis by antagonizing the activity of the anti-apoptotic Bcl-2 family members [14]. In lymphocytes, Bim has been shown to be a major transducer of several apoptotic signals including microtubule destabilization [18]. In this study, we show that 7-ketocholesterol induced, via the calcium-sensitive tyrosine kinase PYK 2 ⁄ CAKb ⁄ RAFTK ⁄ CADTK, a calcium-dependent activa- tion of the MEK 1 ⁄ 2 fi ERK 1 ⁄ 2 survival pathway delaying several apoptotic mechanisms initiated by the oxysterol. We also demonstrate the involvement of Bim in 7-ketocholesterol-induced cytotoxicity. Indeed, we show that Ca 2+ influx leads to the translocation of the protein from the microtubule dynein motor complex to mitochondria, and thus its interaction with Bcl-2 [19]. Therefore, 7-ketocholesterol-induced apoptosis appears to be a complex phenomenon involving several calcium- dependent transduction pathways. Results ERK 1 ⁄ 2 is activated during the first steps of 7-ketocholesterol-induced apoptosis The effects of 7-ketocholesterol were examined in rela- tion to the expression of various signalling proteins in THP-1 cells. Exposure of cells to 7-ketocholesterol resulted in a rapid phosphorylation of ERK 1 ⁄ 2 within 1 h, as monitored through the use of phospho-specific antibodies (Fig. 1A). ERK phosphorylation peaked at 2–3 h, where phosphoERK reached 30% of total ERK vs. 13% in control, and then declined back towards basal levels at 12 h after exposure to 7-ketocholesterol (Fig. 1A,B), whereas apoptosis increased significantly (Fig. 1D). No activation of p38 MAPK or JNK was observed (data not shown) and dephosphorylation of PKB at threonine 308 was noted (Fig. 1C). This PKB dephosphorylation, leading to its inactivation, appeared as soon as 3 h after 7-ketocholesterol treat- ment. However, no phosphorylation of PKB was observed at serine 473 in either control cells or treated 7-keto A B C D P Erk1/2 Erk1/2 7-keto Ctrl 1h 2h 3h 6h 12h 18h Ctrl 1h 2h 3h 6h 12h 18h 300 200 100 150 250 50 0 Ctrl Arbitrary Unit P Erk1/2 protein 1h 2h 3h 6h 12h 18h * * * * * Time of 7-keto treatment P PKB (Thr 308) % of Apoptotic Cells Ctrl 7k20 80 70 60 50 40 30 20 10 0 0 6 12 18 24 30 36 42 48 Time (h) % condensed/fragmented nuclei PKB Fig. 1. 7-Ketocholesterol induces ERK activation and Akt ⁄ PKB inac- tivation. THP-1 cells were treated with 7-ketocholesterol (7-keto, 40 lgÆmL )1 ) for various incubation times. Cell extracts were collec- ted, subjected to SDS ⁄ PAGE and immunoblotted with ERK 1 ⁄ 2, phospho-ERK 1 ⁄ 2, Akt and phospho-Akt thr308 antibodies. Repre- sentative western blots of ERK 1 ⁄ 2–phospho-ERK 1 ⁄ 2 and Akt– phospho-Akt are shown (A and C, respectively); (B) phospho-ERK blot densitometry analysis. Values are means ± SD (n ¼ 3). *P<0.05 vs. control group. (D) Microscopic quantification of cells with fragmented and ⁄ or condensed nuclei was performed using Hoechst 33342 and the percentage of apoptotic cells was deter- mined. Data are means ± SD (n > 5). Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al. 3094 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS cells (data not shown). As ERK 1 ⁄ 2 activation is known to be involved in survival pathways [20], we then focused our attention on the role of the ERK pathway, and looked for the possibility of a relation- ship between ERK 1 ⁄ 2 activation and apoptosis induced by 7-ketocholesterol. Inhibition of the MEK fi ERK pathway accelerates 7-ketocholesterol-induced THP-1 apoptosis To investigate the exact role of ERK in 7-ketocholes- terol-induced apoptosis, we used the MEK inhibitor U0126. Treatment of THP-1 cells with U0126 resulted in the inactivation of ERK 1 ⁄ 2, almost disappearance of the phospho-ERK spot, in agreement with the inhi- bition of MEK, on control cells and at both 3 and 6 h after addition of 7-ketocholesterol (Fig. 2A,B). In light of these results, we investigated the effect of the inhibi- tion of the MEK ERK 1 ⁄ 2 pathway on THP-1 cell viability after 7-ketocholesterol treatment. Whereas U0126 alone has no effect on cell viability, we found that the addition of U0126 accelerates 7-ketocholesterol- induced apoptosis, as shown by the number of apop- totic cells (Fig. 2C). Indeed, apoptosis of THP-1 cells is observed 6 h earlier when cotreated with U0126 and 7-ketocholesterol than when treated with 7-keto- cholesterol alone. This difference of 6 h correlates with the transient activation of ERK 1 ⁄ 2. Thus 7-ketocho- lesterol, which induces THP-1 apoptosis, also activates an anti-apoptotic pathway. Calcium-dependent activation of ERK 1 ⁄ 2 As calcium signals could be involved in MAPK activa- tion and as we and others have previously described a calcium influx in oxysterol-induced apoptosis [8,21,22], we tested whether or not calcium could mediate ERK 1 ⁄ 2 phosphorylation. The role of calcium in MAPK activation was investigated by the cotreatment of THP-1 with 7-ketocholesterol and verapamil, a cal- cium channel blocker which has been described as a potential inhibitor of oxysterol-induced apoptosis. Under these conditions, verapamil inhibited ERK acti- vation early as 3 h after treatment with 7-ketocholes- terol (Fig. 3A,B). These results were strengthened by the use of the intracellular calcium chelator BAPTA which also completly inhibited ERK activation (data not shown). Next, we wondered how calcium could activate the ERK 1 ⁄ 2 pathway. As the cytosolic calcium-dependent PYK 2, a Src kinase activator, could mediate the acti- vation of the Ras-Raf-Mek-Erk pathway [12,23,24], activation by phosphorylation of tyrosines 579⁄ 580 in PYK 2 was investigated using a phosphorylation site- specific antibody. Figure 3C,D shows that PYK 2 was phosphorylated at tyrosines 579 ⁄ 580 as early as 1 h after the THP-1 cells were treated with 7-ketochole- sterol. This phosphorylation peaked at 2 h and then declined back toward basal level at 12 h after the addition of 7-ketocholesterol. Phosphorylated PYK 2 peaked around 56% of total PYK 2 at 2 h vs. 17% in control. However, only weak phosphorylation (15% of PYK phosphorylated as in controls) was detected on these tyrosine residues when cells were cotreated with 7-ketocholesterol and verapamil (Fig. 3E,F) suggesting that calcium is responsible for the activation of PYK 2 as previously demonstrated by Lev et al. [13]. Taken together, these results suggest that calcium uptake activates ERK 1 ⁄ 2 via the activation of PYK 2. 7-keto A B C P Erk1/2 % of Apoptotic Cells Erk1/2 Ctrl U 3h 3h+U 6h 6h+U Arbitrary Unit P Erk1/2 protein 250 150 200 100 50 0 Ctrl Ctrl U U 3h 6h3h+U 7k20 7k20 + U * * * * * * 45 35 25 15 5 0 0 %condensed/fragmented nuclei 6121824 Time (h) 40 30 20 10 6h+U Time of 7-keto treatment Fig. 2. The MEK blocker U0126 inhibits 7-ketocholesterol-induced ERK activation and accelerates apoptosis. THP-1 cells were either untreated (Ctrl) or incubated with U0126 (U, 10 lmolÆL )1 ) alone or in association with 7-ketocholesterol (7-keto) for the indicated times. (A) Cell lysates were subjected to SDS ⁄ PAGE, and immuno- blot analysis with antibodies against ERK 1 ⁄ 2 or phospho-ERK 1 ⁄ 2 was performed. (B) Phospho-ERK blot densitometry analysis. Val- ues are means ± SD (n ¼ 3). (C) Microscopic quantification of cells with fragmented and ⁄ or condensed nuclei was performed using Hoechst 33342 and the percentage of apoptotic cells was deter- mined. Data are means ± SD (n ¼ 4) (*P < 0.05). A. Berthier et al. Pathways in 7-ketocholesterol-induced apoptosis FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3095 ERK 1/2 inhibits 7-ketocholesterol-induced apoptosis by phosphorylation of BAD Having established the role of ERK 1 ⁄ 2 in the 7-keto- cholesterol survival pathway, we wondered how ERK inhibited cell death. During apoptosis involving mito- chondria, Bcl-2 family members play a critical role. We have previously described that 7-ketocholesterol induced BAD dephosphorylation at serines 75 and 99, but BAD dephosphorylation at serine 75 was incom- plete [8]. Moreover, Scheid et al. [11] showed that ERK could phosphorylate BAD at serine 75, via p90 RSK .We therefore tested the hypothesis that ERK could reduce apoptosis by phosphorylating BAD. To characterize the importance of the ERK pathway in the activa- tion ⁄ inhibition of BAD during the early steps of 7-ketocholesterol-induced apoptosis, the status of BAD phosphorylation at serine 75 was investigated following treatment with the MEK inhibitor U0126 (Fig. 4A,B). Western blot analysis revealed that BAD was only slightly dephosphorylated at serine 75 at 12 h of treat- ment with 7-ketocholesterol alone (a 15–20% decrease of phosphorylated BAD), whereas cotreatment of THP-1 with the oxysterol and U0126 induced the same rate of dephosphorylation of BAD as early as 3 h. We next asked whether the acceleration of BAD dephosphorylation induced by U0126 could have an impact on mitochondria integrity. Cells were pretreated with U0126, followed by various 7-ketocholesterol treatments, and the transmembrane mitochondrial potential was measured (Fig. 4C). In the absence of a MEK inhibitor, mitochondria depolarization first appeared no later than 12 h after the beginning of treatment, whereas in the presence of U0126, the trans- membrane mitochondrial potential declined as early as 6 h. We next wondered whether U0126 could also accelerate cytochrome c and Smac ⁄ Diablo leakage from the mitochondria. Western blot analysis was per- formed and we showed that U0126 increased the release of cytochome c and Smac ⁄ Diablo induced by 7-ketocholesterol treatment (Fig. 4D). Taken together, these results indicate that BAD is phosphorylated at ser75 after ERK activation by 7-ketocholesterol and this phosphorylation delays apoptosis by preventing mitochondrial damage. Secondary decreases of PYK 2 and ERK 1 ⁄ 2 acti- vities do not appear to be related to a decrease in cytoplasmic calcium. As we previously described that 7-ketocholesterol treatment of THP-1 cells induced an increase of cyto- plasmic free calcium [8], a time course of intracellular calcium fluctuations was performed between addition of 7-ketocholesterol and appearance of a significant number of apoptotic cells to investigate the relation- ship between changes in intracellular calcium, activa- tion of PYK 2-ERK 1 ⁄ 2 pathway and apoptosis. As shown in Fig. 5 calcium concentration increased until 12 h when, whereas PYK 2 and ERK 1 ⁄ 2 activities peaked at 2–3 h as shown above. So the secondary decrease of PYK 2 and ERK 1 ⁄ 2 activities did not appear to be related to a decrease in cytoplasmic free calcium. 7-keto A B C D E F 7-keto P Erk1/2 Erk1/2 P PYK2 PYK2 P PYK2 PYK2 Arbitrary Unit 200 Ctrl Ctrl 1h 2h 3h 6h 12h 18h 7-keto Ctrl 1h 2h 3h 6h 12h 18h Vera 3h 6h 6h+Vera3h+Vera Ctrl Vera 3h 6h 6h+Vera3h+Vera Ctrl Vera 3h 6h 6h+Vera3h+Vera Ctrl Vera 3h 6h 6h+Vera Time of 7-keto treatment Time of 7-keto treatment Time of 7-keto treatment * * * * 3h+Vera 100 150 50 0 P Erk1/2 protein Arbitrary Unit 200 120 160 80 40 0 P PYK2 protein Arbitrary Unit 200 100 150 50 0 P PYK2 protein Fig. 3. 7-Ketocholesterol-induced calcium influx activates ERK phos- phorylation through PYK 2. THP-1 cells were either untreated (Ctrl) or incubated with L-type calcium channel blocker, verapamil (Vera, 100 lmolÆL )1 ) alone or in association with 7-ketocholesterol for the indicated times. Cell extracts were collected, subjected to SDS ⁄ PAGE and immunoblotted with ERK 1 ⁄ 2 and phospho- ERK 1 ⁄ 2 (A), or PYK 2 and phospho-PYK 2 (C, E) antibodies. (B, D, F) Respective group densitometry results. Values are means ± SD (n ¼ 3). *P<0.05 vs. control group. Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al. 3096 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS Bim activation depends on calcium uptake Besides BAD, Bim functions also as a sensor toward apoptotic stimuli by heterodimerization and inactiva- tion of anti-apoptotic multi-BH domain proteins. Moreover, it has been shown that Bim could be under control of the MEK fi ERK pathway [25]. Indeed, ERK 1 ⁄ 2 is known to exert its anti-apoptotic activity in promoting phosphorylation and consequently the proteasome-dependent degradation of Bim [26]. Inter- estingly, in THP-1 cells, Bim EL is expressed in control cells and the level of Bim EL , as well as Bcl-2, is not apparently changed by 7-ketocholesterol treatment (Fig. 6A). Moreover, the use of the ERK inhibitor, U0126, does not affect Bim EL or Bcl-2 expression, sug- gesting that the transient activation of ERK 1 ⁄ 2by Calcium Influx Ctrl Vera 7-keto 7-keto + Vera 0 2h 4h 6h 8h 10h 12h Time of 7-keto treatment (Hours) 70 60 50 40 30 20 10 0 Fluo-3 Fluorescence Arbitrary Unit Fig. 5. Time course of intracellular calcium after 7-ketocholesterol treatment of THP-1 cells. THP-1 cells were incubated in the pres- ence of 7-ketocholesterol (40 lgÆmL )1 , 7-keto), with or without verapamil (100 lmolÆL )1 , Vera) or in the absence of oxysterol (Ctrl). After different incubation times, cells were loaded with fluo-3 ⁄ AM and the dye fluorescence was measured by flow cytometry. Each fluorescent data point is normalized to the maximal fluo-3 fluores- cence induced in cells treated with ionomycin (2 lmolÆL )1 ). Data are the means ± SD. P Bad (S75) Bad Hsc 70 Ctrl 800 600 400 200 0 U 3h 6h 12h 12h+U3h+U 7-keto A B C D % of Cells with Depolarized Mitochondria Smac / Diablo S100 Fraction Cytochrome c Hsc 70 Time (h) % DiOC 6 (3) negative cells 6h+U Ctrl U 18h 24h 30h18h+U 24h+U 30h+U 7-keto Ctrl U 7K20 7K20 + U 70 60 50 40 30 20 10 0 0 6 12 18 24 3h 6h 12h 12h+U Time of 7-keto treatment * * † * * * * * † † 3h+U 6h+U Arbitrary Unit P Bad protein Fig. 4. 7-Ketocholesterol-induced ERK phosphorylation inhibits BAD dephosphorylation, mitochondria depolarization and cytochrome c–Smac ⁄ Diablo release. (A) Western blot analysis of BAD and phospho-BAD was performed during 7-ketocholesterol (7-keto) and U0126 (U) treatment of THP-1 cells. (B) Phospho-BAD blot densi- tometry analysis. Values are means ± SD (n ¼ 3). *P<0.05 vs. Ctrl or U group; P<0.05 vs. Ctrl or oxysterol-treated cells. (C) Transmembrane mitochondrial potential was measured by flow cytometry using DiOC 6 (3) dye. After the incubation period, fluores- cence associated with DiOC 6 (3) was measured by flow cytometry and 10 000 cells were analysed for each assay. Results represent the means ± SD (n ¼ 4) (*P<0.05). (D) THP-1 cells were either untreated (Ctrl), treated with U0126 (U) or with 7-ketocholesterol (7-keto) for 18, 24 or 30 h alone or in association with U0126, and cytosol fractions (S100 fraction) were collected. Subcellular frac- tions were subject to SDS ⁄ PAGE and immunoblot analyses were performed with antibodies against cytochrome c or Smac ⁄ Diablo. Hsc70 was used as internal control loading. The blots are represen- tative of two independent experiments. 7-ketoA B 7-keto Ctrl Ctrl Bim EL Bcl-2 Bim EL Bcl-2 U3h 6h3h+U 6h+U 1h 2h 3h 6h 12h 18h Fig. 6. 7-Ketocholesterol and ERK activation do not regulate Bim expression. THP-1 cells were either untreated (Ctrl), treated with U0126 (U) or with 7-ketocholesterol (7-keto) alone or in association with U0126 for the indicated period, and cell extracts were collec- ted. Lysates were analysed by western blotting using Bim and Bcl-2 antibodies (A, B). A. Berthier et al. Pathways in 7-ketocholesterol-induced apoptosis FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3097 7-ketocholesterol does not induce the degradation of Bim EL in THP-1 cells (Fig. 6B). In light of these results, we wondered if Bim EL was involved in 7-ketocholesterol-induced apoptosis, because BAD does not seem to be the only pro-apoptotic molecule involved [27]. Indeed, Puthalakath et al. [19] described that the pro-apoptotic activity of Bim can also be regulated by interaction with the dynein motor complex and the microtubules. As we previously described that 7-ketocholesterol induced a calcium uptake and as it is known that calcium uptake could destabilize microtubules [28] dissociating Bim from microtubule dynein motor complex, we wondered if 7-ketocholesterol-induced calcium influx could modify microtubule structure and Bim EL localization. Thus, we examined the effects of 7-ketocholesterol, alone or in association with verapamil, on microtubule organ- ization using classical fluorescence microscopy. Both control cells and verapamil treated cells displayed a typical randomly oriented, intact microtubular network (Fig. 7A,B). In 7-ketocholesterol-treated cells, a pro- gressive disorganization ⁄ reorganization of microtubules occurred. These modifications were time-dependent and affected more than 90% of the cells (Fig. 7C,E and data not shown). This reorganization ⁄ disorganization was partially prevented in the presence of verapamil tubulin α α Hoechst 33342 Overlay Ctrl A B C D E F Vera 7-keto 6h 7-keto 6h + 7-keto 12h 7-keto 12h + Vera Vera Fig. 7. Analysis of calcium involvement in 7-ketocholesterol-induced THP-1-microtubule- network disruption. Cells were analysed by indirect immunofluorescence microscopy using anti-(a-tubulin) Igs and Hoechst 33342 as described in Experimental procedures showing microtubules and nuclear structure of (A) untreated cells (Ctrl) (B) verapamil- treated cells (Vera), 7-ketocholesterol-trea- ted cells alone (C, E) or in association with verapamil (D, F). Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al. 3098 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS (Fig. 7D,F), suggesting that calcium uptake could be the event that initiates microtubule destabilization. Thus, we wondered if inactivated Bim is targeted to microtubules and if 7-ketocholesterol treatment of THP-1 cells could change its localization. Co-immuno- precipitation experiments revealed that in untreated cells Bim EL is complexed with microtubules and not with Bcl-2, whereas as 7-ketocholesterol treatment time progresses, Bim EL colocalized with Bcl-2 and not with microtubules, the part of Bcl2 in anti-Bim immunoprecipitate (IP Bim) growing from 18% in control to 50% after 24 h of treatment, a 2.7-fold increase. (Fig. 8A,B). Moreover, cotreatment of the cells with 7-ketocholesterol and verapamil appears to abolish the dissociation of Bim EL from the microtu- bules and consequently its translocation to Bcl-2, the part of Bcl2 in IP Bim is only of 24% after 24 h of cotreatment with verapamil. We next investigated the localization of Bim EL at the mitochondrial level under 7-ketocholesterol treatment alone or in association with verapamil. The results from this investigation confirmed the increase of Bim EL levels in the mito- chondria and that this increase is inhibited by the presence of verapamil (Fig. 8C). So, Bim EL relocaliza- tion at the mitochondrial level is dependent on cal- cium influx induced by 7-ketocholesterol. Discussion In a prior report [8], we demonstrated that a major step in the apoptotic response to 7-ketocholesterol of human monocytic cell line THP-1 is the sustained influx of extracellular Ca 2+ leading to the dephospho- rylation of the pro-apoptotic ‘BH3-only’ protein BAD. Moreover we demonstrated that this dephosphoryla- tion was mediated by calcineurin (PP2B). However, this dephosphorylation was incomplete and occurred more quickly at serine 99 than at serine 75, suggesting the existence of alternative mechanisms leading to apopto- sis. Moreover, this idea was confirmed by the work of Panini et al. describing that calcium-dependent activa- tion of cPLA2 led to the stimulation of arachidonate release and to apoptosis [29]. However, the lack of a complete inhibition of apoptosis in cPLA2 (– ⁄ –) macro- phages also led this group to point out the existence of other pathways regulating oxysterol-induced cell death. Therefore, oxysterol-induced apoptosis appears to be a complex phenomenon with multiple initiation pathways and several apoptotic mechanisms. In this study, we examined two paradoxical effects induced by 7-ketocholesterol in THP-1 cells. Indeed, our results demonstrate a calcium-dependent activation of one survival pathway, the PYK 2 fi MEK 1 ⁄ 2 fi IP Bim A B C 7-keto 7-keto Ctrl Vera 6h 6h +Vera Tubulin α tubulin 500 400 300 200 100 0 Arbitrary Unit Ctrl Vera 6h 6h+Vera 12h+Vera 18h+Vera 24h+Ve ra 12h 18h 24h Bcl-2 Time of 7-keto treatment 7-keto Ctrl 6h 12h 18h WB : Bim - Vera + Vera Mitochondria * * * * † † † † Bcl-2 Bim EL +Vera +Vera +Vera 12h 12h 18h 18h 24h Ctrl 6h 12h 18h 24h 24h Total Extract Fig. 8. 7-Ketocholesterol-induced calcium influx activates the dissociation of Bim from microtubules and consequently its transloca- tion to Bcl-2 at the mitochondrial level. (A) THP-1 cells were untreated (Ctrl), treated with verapamil (Vera) or with 7-ketocholes- terol alone (7-keto) or in association with verapamil for 6, 12, 18 or 24 h. After treatment, anti-Bim immunoprecipitates were collected, subjected to SDS ⁄ PAGE, and immunoblotted with antibodies specific for a-tubulin, Bcl-2 or Bim. Western blots were also performed on total extract to check the levels of a-tubulin, Bcl-2 or Bim. (B) Tubulin and Bcl-2 blot densitometry analysis. Values are means ± SD, (n ¼ 3). *P<0.05 vs. Ctrl or Vera group; P<0.05 vs. Ctrl or oxysterol-treated cells. (C) THP-1 cells were untreated (Ctrl), treated with verapamil (Vera) or with 7-ketocholesterol alone (7-keto) or in association with verapa- mil for 6, 12 or 18 h. Mitochondrial fractions were collected and subjected to western blot analysis with antibodies against Bim. The blots are representative of three independent experiments. A. Berthier et al. Pathways in 7-ketocholesterol-induced apoptosis FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3099 ERK 1 ⁄ 2 pathway allowing BAD phosphorylation on serine 75, as well as one additional apoptotic pathway inducing the translocation of Bim from microtubules to Bcl-2 at the mitochondrial level, which leads to mit- ochondrial damage and apoptosis. In fact, the use of U0126, a MEK 1 ⁄ 2 inhibitor which accelerates 7-keto- cholesterol-induced THP-1 apoptosis, clearly suggests that this signalling pathway acts as a survival pathway. In most, but not all systems studied, activation of MEK 1 ⁄ 2 and ERK 1⁄ 2 is associated with the inhibi- tion of cell death [30,31]. The mechanism by which this occurs is not completely understood, but could be rela- ted to the phosphorylation of BAD, and thus, to its inactivation [11]. Moreover, direct anti-apoptotic actions of ERK 1 ⁄ 2 related to the phosphoryla- tion ⁄ inactivation of caspase-9 [32] and to the phos- phorylation ⁄ degradation of Bim [26] have been recently described. It is therefore surprising that expo- sure of leukaemic cells to 7-ketocholesterol at concen- trations that trigger apoptosis was associated with the clear activation of ERK 1 ⁄ 2, even if it is a transient phenomenon. Moreover, it seems that other MAPKs (JNK or p38 MAPK) were not activated under 7-keto- cholesterol treatment of THP-1 cells, suggesting that ERK activation is not under the activation of other MAPKs as described by Numazawa et al. [33]. Con- trary to Rusin ˜ ol et al. [27], who described Akt ⁄ PKB degradation under oxysterol treatment of macrophage cells, we never noticed a degradation of Akt ⁄ PKB in THP-1 cells during the course of 7-ketocholesterol treatment but we did show that Akt ⁄ PKB was inacti- vated by dephosphorylation at threonine 308. As we previously described a calcium influx in 7-ketocholesterol-induced THP-1 apoptosis, we exam- ined the role of calcium in ERK 1 ⁄ 2 activation. Hence, the L-Type calcium channel blocker verapamil and intracellular calcium chelator BAPTA completely inhibited 7-ketocholesterol-induced ERK activation. Interestingly, calcium-dependent activation of the MEK 1 ⁄ 2 fi ERK 1 ⁄ 2 pathway has been previously described in 7b-hydroxycholesterol-treated aortic smooth muscle cells [21]. Nevertheless, the authors did not describe the effects or the mechanism of ERK activation. Protein tyrosine kinases (PTKs) transduce key extracellular signals that trigger various biological events, such as cytoskeletal rearrangement and mitogenesis. Among the PTKs, PYK 2 (also known as CAKb, RAFTK or CADTK), which exists mainly in the cytoplasm [34], is abundantly expressed in haemopoeitic cells and in the brain. Moreover, PYK 2 is activated by stimuli that increase the concentrations of intracellular Ca 2+ . Indeed elevation of intracellular calcium triggers activation of PYK 2 as described by Lev et al. [13] and a maximal cata- lytic activity was observed after phosphorylation of PYK 2 at tyrosines 579 and 580 in the kinase domain activation loop [35]. Thus, in untreated THP-1 cells, as described by Yamasaki et al. [36], PYK 2 was poorly phosphorylated at tyrosines 579 ⁄ 580, but after 7-ketocholesterol treatment we saw an increase of its phosphorylation. Moreover, our data demonstrate that verapamil inhibits 7-ketocholesterol-mediated- PYK 2 phosphorylation, suggesting the role of this protein tyrosine kinase in ERK activation. However the secondary decreases of PYK 2 and ERK1 ⁄ 2 acti- vites are not related to a decrease of cytoplasmic free calcium suggesting that 7-ketocholesterol treatment of THP-1 induces others transduction pathways leading to a secondary inactivation of these kinases. We next examined the ability of the MAPK signal- ling pathway to inhibit, via p90 RSK , the apoptotic effect of 7-ketocholesterol by phosphorylating BAD. Treatment of cells with the combination of the MEK inhibitor U0126 and 7-ketocholesterol caused an ear- lier dephosphophorylation of BAD than with the oxy- sterol alone. The induction of cell death was also faster following cotreatment than treatment with 7-ketocholesterol alone, indicating that inhibition of BAD phosphorylation at serine 75 increases apoptosis. This process could accelerate the disruption of the mito- chondrial transmembrane potential and Smac ⁄ diablo and cytochrome c release into the cytosol. Hence, our findings suggest that the MAPK signalling pathway pro- motes cell survival by a mechanism that modulates the cell death machinery directly by phosphorylating and thereby inactivating the pro-apoptotic protein BAD. Our previous results also suggest the possibility of the involvement of other Ca 2+ -initiated cell death pathways. Therefore our results showing the release of the pro-apoptotic ‘BH3 only’ protein Bim and its association with Bcl-2 in a calcium-dependent man- ner complement our knowledge on the mechanism of 7-ketocholesterol-induced apoptosis. A calcium- dependent destabilization of microtubules, as previ- ously described by Keith et al. [33], probably induces the dissociation of Bim from the microtubule dynein motor complex, allowing inactivation of anti-apopto- tic, multi-BH domain proteins such as Bcl-2 or Bcl-X L in the mitochondria. Previously Palladini et al. des- cribed a 7-ketocholesterol induced destabilization of vimentin filament architecture without significant alter- ations of the microtubule network on a bovine aortic endothelial cell line. Nevertheless they showed that 7-ketocholesterol induced tubulin aggregates and so they did not exclude a possible role of microtubules in oxysterol-induced endothelial cell apoptosis [37]. It has Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al. 3100 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS been reported that activation of the ERK 1 ⁄ 2 signaling pathway promoted phosphorylation and proteasome- dependent degradation of Bim, but this result was not obtained in our study. The possible explanation could be that the length and intensity of activation of ERK 1 ⁄ 2 is not sufficient to induce a detectable change in Bim concentration. The short length of ERK activation and the dephosphorylation of Akt ⁄ PKB could be related to the activation of phosphatases such as protein phosphatase 1a [38]. Hence, 7-keto- cholesterol-induced apoptosis appears to be a com- plex phenomenom implicating several transduction pathways, two pro-apoptotics and surprisingly one anti-apoptotic, that to our knowledge have not been previously described. However further studies will be necessary to try to quantify the part of these different pathways in 7-ketocholesterol induced cytotoxicity. All of these mechanisms seem to be related to the sus- tained rise of free cytosolic calcium triggered by the transfer of Trpc-1 into lipid raft domains that we have previously described [8] and involve the mitochondrial pathway of apoptosis with the implication of the pro- apoptotic BH3 proteins BAD and Bim. As all oxysterols are not able to induce apoptosis and as cytotoxicity can vary according cell type, it will be interesting to use other oxysterols and other cell types and especially other cell lines of the monocytes ⁄ macro- phages lineage to investigate the implication of these different pathways and particularly sustained calcium ion influx, which appears to be the key event in oxy- sterol-mediated cytotoxicity. In this way the first experi- ments performed by our group on U937, another monocytic cell line, with 7-ketocholesterol (A. Berthier, unpublished results) produced results quite similar to those obtained with THP-1 cells. Experimental procedures Reagents and antibodies The THP-1 human monocytic cell line was from the Ameri- can Tissue and Culture Collection (Manassas, VA, USA). DiOC 6 (3) and the anti-mouse IgG-AlexaFluor 565 were from Molecular Probes, Inc. (Eugene, OR, USA). 7-Keto- cholesterol, IgePal, EGTA, Pipes, MgSO 4 , mannitol, dimethylsulfoxide, verapamil, pepstatin A, aprotinin, tryp- sine inhibitor, leupeptin, phenylmethylsulfonylfluoride, paraformaldehyde, Hoechst 33342, NaF, b-glycerophos- phate and Triton X-100 were from Sigma (Sigma-Aldrich, L’Isles d’Abeau-Chesnes, France). The anti-Smac ⁄ Diablo polyclonal antibody was from Imgenex (San Diego, CA, USA). The anti-BAD monoclonal antibody and the anti-BAD phospho Ser112 (human Ser75) polyclonal anti- body were from Upstate Biotechnology (Lake Placid, NY, USA), and the anti-cytochrome c monoclonal antibody was from Pharmingen (San Diego, CA, USA). The anti-(Hsc 70), anti-PYK 2, anti-PYK 2 phospho tyr279 ⁄ tyr580 and Bim polyclonal antibodies, the anti-(a-tubulin) monoclonal antibody, protein G-Agarose, and total mouse IgG were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), the anti-Erk 1 ⁄ 2, anti-Erk 1 ⁄ 2 phospho Thr202 ⁄ Tyr204, anti-PKB and anti-PKB phospho Thr308 and U0126 were from Cell Signaling Technology (Cell Signaling Technology, Hitchin, UK) and the anti-Bcl-2 Ig was from Dako (Dako, Trappes, France). Cell culture Human monocytic THP-1 cells were grown in RPMI 1640 with glutamax-I (Gibco, Eragny, France) and antibiotics (100 UÆmL )1 penicillin, 100 lgÆmL )1 streptomycin) (Gibco), supplemented with 10% (v ⁄ v) heat-inactivated fetal bovine serum (Gibco). The cells were incubated at 37 °C under a 5% CO 2 ⁄ 95% air atmosphere (v ⁄ v). Cell treatment For all experiments, a 7-ketocholesterol stock solution was prepared at a concentration of 800 lgÆmL )1 as previously described [39]. 7-Ketocholesterol was added to the culture medium for a final concentration of 40 lgÆmL )1 . This con- centration is in the range of levels measured in human plasma after a meal rich in fat [40]. Verapamil, an L-type calcium channel inhibitor, was added to the culture medium at a final concentration of 100 lmolÆL )1 . The MEK fi Erk inhibitor U0126 was used at a final concentration of 10 lmolÆL )1 .In all experiments, verapamil and U0126 were introduced in the culture medium 30 min before 7-ketocholesterol. Characterization of nuclear morphology by staining with Hoechst 33342 Nuclear morphology of control and treated cells was studied by fluorescence microscopy after staining with Hoechst 33342 (kEx max , 346 nm; kEm max , 420 nm) used at 10 lgÆmL )1 . The morphological aspect of cell deposits, applied to glass slides by cytocentrifugation with a cytospin 4 centrifuge (Shandon, UK), was observed with an Axioskop light micro- scope (Zeiss, Jena, Germany) by using UV light excitation. Three hundred cells were examined for each sample. Flow cytometric measurement of mitochondrial transmembrane potential ( DW m ) with the dye DiOC 6 (3) Variations of the mitochondrial transmembrane potential (DY m ) were measured with 3,3¢-dihexyloxacarbocyanine A. Berthier et al. Pathways in 7-ketocholesterol-induced apoptosis FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS 3101 iodide (DiOC 6 (3); kEx max : 484 nm, kEm max : 501 nm) used at a final concentration of 40 nmolÆL )1 [41]. The flow cyto- metric analyses were performed on a Galaxy flow cytometer (Partec, Munster, Germany) and the green fluorescence was collected through a 524 ⁄ 44-nm band pass filter. Fluorescent signals were measured on a logarithmic scale of four dec- ades of log. For each sample, 10 000 cells were acquired and the data were analysed with flomax software (Partec). Flow cytometric measurement of cytosolic calcium with the dye Fluo-3 THP-1 cells were washed with NaCl ⁄ P i (pH 7.4) and then incubated with Fluo-3 ⁄ AM (6 lmolÆL )1 ; kEx max : 506 nm, kEm max : 526 nm) for 30 min at 37 °C in Hank’s balanced salt buffer (pH 7.2) with Pluronic F-127. After loading, cells were suspended in Hepes buffer (pH 7.4) supplemented with probenecid (5 mm) to prevent leakage of the dye. Fluorescence was measured by flow cytometry with a Gal- axy flow cytometer (Partec) using a 524 ⁄ 44-nm band pass filter. For each sample, events were acquired for 60 s and the data were analysed with flomax software (Partec). Staining for microtubules Treated or control cells were rinsed twice in microtubule- stabilizing buffer (MSB; 50 mm Pipes pH 6.9, 10 mm EGTA, and 10 mm MgSO4). Cells were immediately fixed for 1 h in MSB containing 4% (w ⁄ v) freshly prepared para- formaldehyde and 0.2 mm mannitol. Cells were then washed twice with MSB, fixed on polylysine glass and per- meabilized for 20 min with 0.5% (w ⁄ v) Triton X-100 in MSB containing 0.2 molÆL )1 mannitol. After two washes in MSB, cells were treated for 2 h at room temperature with 5% (w ⁄ v) BSA in MSB to block nonspecific antibody bind- ing sites. Cells were then incubated overnight at 4 °C in the presence of the primary mouse monoclonal antibody against a-tubulin [1 : 100 dilution in MSB containing 0.1% (v ⁄ v) MSB] and rinsed twice in MSB containing 0.1% (w ⁄ v) BSA. Cells were then incubated for 1 h in the pres- ence of the secondary AlexaFluor 565-conjugated rabbit anti-mouse antibody (1 : 250 dilution in MSB containing 0.1% MSB). After two washes in MSB, cells were mounted in Fluoprep. At least 50 cells were examined for each experiment and three independent experiments were per- formed for each treatment. Observations were performed on an Axioskop light microscope (Zeiss, Jena, Germany) by using UV light excitation. Immunoprecipitation and western blotting For the immunoprecipitation of the Bim protein, cells were suspended in immunoprecipitation buffer [10 mm Tris ⁄ HCl, 140 mm NaCl, 0.1% (w ⁄ v) IgePal] containing a mixture of protease inhibitors (0.1 mm phenylmethanesulfonyl fluoride, 2.5 lgÆL )1 aprotinin, 10 l g ÆL )1 pepstatin A, 2.5 lgÆL )1 trypsin inhibitor and 2.5 lgÆL )1 leupeptin). After a 20-min incubation at 4 °C in the lysis buffer, the cell debris were eliminated by centrifugation for 10 min at 10 000 g. The resulting supernatant was precleared by adding 1 lg total mouse IgG and 50 lL protein G-agarose for 30 min. After a 10 000 g centrifugation (4 °C, 10 min), the supernatant was collected, adjusted to 500 lL in lysis buffer and incu- bated overnight at 4 °C with 10 lg of the anti-Bim Ig on a rotating device. After the incubation period, 50 lL protein G-agarose were added and the sample was incubated for 2 h before collecting the immunoprecipitates by centrifuga- tion at 10 000 g (4 °C, 5 min). After washing the pellet four times, the immunoprecipitation extract was suspended in Laemmli’s buffer [1% (w ⁄ v) SDS, 1 mm sodium orthovana- date, 10 mm Tris ⁄ HCl]. Alternatively, cells were resuspended in Ripa lysis buffer [0.1% (w ⁄ v) SDS, 1% (w ⁄ v) IgePal, 0.5% (w ⁄ v) Na-deso- xycholate, 50 mm Tris ⁄ Hcl pH 8.0, 150 mm NaCl] contain- ing a mixture of protease and phosphatase inhibitors (0.1 mm phenylmethanesulfonyl fluoride, 2.5 lgÆL )1 aproti- nin, 10 lgÆL )1 pepstatin A, 2.5 lgÆL )1 trypsin inhibitor, 2.5 lgÆL )1 leupeptin, 0.1 mm Na-orthovanadate, 40 mm b-glycerophosphate, 100 mm NaF). After a 30-min incuba- tion at 4 °C in the lysis buffer, the cell debris were eliminated by centrifugation for 20 min at 10 000 g and the supernatant was collected. The release of cytochrome c and Smac ⁄ Diablo from mito- chondria to the cytosol was investigated by western blot ana- lysis of THP-1 cells incubated for 18, 24 or 30 h with 7-ketocholesterol alone or in association with U0126 as pre- viously described [42]. Mitochondria were obtained before the last 1000 g centrifugation generating the cytosol fraction. The protein concentrations were measured by using bicinchoninic acid reagent (Pierce, Rockford, IL, USA) according to the method of Smith et al. [43]. Seventy micro- grams of protein were incubated in loading buffer [125 mmolÆL )1 Tris ⁄ HCl, pH ¼ 6.8, 10% (w ⁄ v) 2-merca- ptoethanol, 4.6% (w ⁄ v) SDS, 20% (v ⁄ v) glycerol, 0.003% (w ⁄ v) Bromophenol blue], boiled for 3 min, separated by SDS ⁄ PAGE and electroblotted onto a polyvinylidine diflu- oride membrane (Bio-Rad, Ivry sur Seine, France). After blocking nonspecific binding sites for 2 h at room tempera- ture in TPBS [NaCl ⁄ P i , 0.1% (v ⁄ v) Tween-20], the mem- branes were incubated overnight at 4 °C with the primary antibody diluted in TPBS. After three 10-min washes with TPBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody at a dilution of 1 : 2500 for 1 h at room temperature and washed three times in TPBS for 10 min. Autoradiography of the immu- noblots was performed using an enhanced chemolumines- cence detection kit (Amersham, Les Ulis, France). Western blots were quantified using a JS800 densitometer using Pathways in 7-ketocholesterol-induced apoptosis A. Berthier et al. 3102 FEBS Journal 272 (2005) 3093–3104 ª 2005 FEBS [...]... Berthier et al Pathways in 7-ketocholesterol-induced apoptosis quantity one software (Bio-Rad) Each experiment was repeated three times with identical results 9 Statistical methods Statistical analysis were performed with statview software (Cary, NC, USA) using a two-way analysis of variance followed by Student’s t-test 10 Acknowledgements 11 This work was supported by the University of Bourgo´ gne,... 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