Báo cáo khoa học: Enhanced apoptosis through farnesol inhibition of phospholipase D signal transduction pdf

Enhanced apoptosis through farnesol inhibition of phospholipase D signal transduction Marcia M. Taylor 1 , Kendra MacDonald 1 , Andrew J. Morris 2 and Christopher R. McMaster 1 1 Atlantic Research Centre, Dalhousie University, Halifax, Canada 2 Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, USA Phosphatidylcholine (PC) is the major membrane lipid found in eukaryotic cells, comprising  50% of phos- pholipid mass. PC plays a major role in maintaining the physical properties of membranes and is also a res- ervoir of signaling molecules [1–4]. A major signal transduction pathway initiated from PC is its catabol- ism by phospholipase D (PLD) to yield phosphatidic acid (PA), which can be dephosphorylated by PA phosphatase activity to generate diacylglycerol (DAG) (Fig. 1) [3,5–14]. Both PA and DAG can directly bind to proteins within the cell and modulate numerous cellular events including those that regulate apoptotic life-and-death decisions [2,4,15]. Apoptosis is normally required during development as well as in the removal of adult cells that have reached the end of their normal lifespan. Misregulation of the apoptotic process contri- butes to tumorgenicity and many cancer chemo- therapeutics preferentially induce apoptosis in cancer cells [16]. Farnesol is a natural compound whose exogenous administration has been observed to preferentially cause apoptosis in neoplastic vs. normal cells [17,18]. Farnesol is produced by dephosphorylation of farnesol pyrophosphate, a metabolite of the cholesterol biosyn- thetic pathway [19]. Farnesol pyrophosphate can also be used to donate farnesol for covalent prenylation of proteins and is an essential process for oncogenic Ras to affect cellular tranformation [20,21]. The ability of farnesol administration to alter the prenylation of the small G proteins Ras and Rap1A has been previously tested and neither the prenylation event nor the ability of the G proteins to associate with the membrane was Keywords apoptosis; diacylglycerol; farnesol; phosphatidic acid phosphatase; phospholipase D Correspondence C. McMaster, Atlantic Research Centre, Departments of Pediatrics and Biochemistry & Molecular Biology, Dalhousie University, 5849 University Avenue, Room C302, Halifax, Nova Scotia B3H 4H7, Canada Fax: +1 902 494 1394 Tel: +1 902 494 2953 E-mail: Christopher.mcmaster@dal.ca (Received 15 May 2005, revised 3 August 2005, accepted 11 August 2005) doi:10.1111/j.1742-4658.2005.04914.x Farnesol is a catabolite of the cholesterol biosynthetic pathway that prefer- entially causes apoptosis in tumorigenic cells. Phosphatidylcholine (PC), phosphatidic acid (PA), and diacylglycerol (DAG) were able to prevent induction of apoptosis by farnesol. Primary alcohol inhibition of PC cata- bolism by phospholipase D augmented farnesol-induced apoptosis. Exogen- ous PC was unable to prevent the increase in farnesol-induced apoptosis by primary alcohols, whereas DAG was protective. Farnesol-mediated apopto- sis was prevented by transformation with a plasmid coding for the PA phosphatase LPP3, but not by an inactive LPP3 point mutant. Farnesol did not directly inhibit LPP3 PA phosphatase enzyme activity in an in vitro mixed micelle assay. We propose that farnesol inhibits the action of a DAG pool generated by phospholipase D signal transduction that nor- mally activates an antiapoptotic ⁄ pro-proliferative target. Abbreviations CHO, Chinese hamster ovary; DAG, diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; LPP, lipid phosphate phosphatase; PA, phosphatidic acid; PARP, polyADP-ribose polymerase; PC, phosphatidylcholine; PLD, phospholipase D; RasGEF, Ras guanine exchange factor; TBS, Tris-buffered saline. 5056 FEBS Journal 272 (2005) 5056–5063 ª 2005 FEBS altered [22]. Thus, it appears that farnesol administra- tion preferentially kills transformed cells by a mechan- ism independent of protein prenylation. Previous studies found that the addition of PC, but not other phospholipids, was able to rescue cells from farnesol-mediated apoptosis [23,24]. PC was much less effective at preventing apoptosis induced by campto- thecin, etoposide, or chelerythrine [23] implying that some specificity for farnesol exists. It was hypothesized that farnesol inhibition of cholinephosphotransferase, the final step in PC synthesis, was the apoptotic trigger [22,23] as farnesol-mediated apoptosis could be preven- ted by the exogenous addition of the cholinephospho- transferase substrate DAG, or its product, PC, but not by other lipids [23]. However, restoration of PC syn- thesis to normal levels by increasing the expression of cholinephosphotransferase did not alter the ability of farnesol to cause apoptosis, and the addition of DAG to cells did not prevent farnesol-mediated inhibition of PC synthesis, indicating that inhibition of PC synthesis by farnesol was not the apoptotic trigger [24]. Analysis of the PC metabolic pathways also links PC to DAG through hydrolysis by PLD to produce PA and subsequent dephosphorylation to DAG [2,3,7]. As the generation of PA and DAG by lipid turnover is a major means by which cells regulate cell growth we reasoned that this pathway may be a major contributor to the signal required for farnesol to induce apoptosis. Results Inhibition of PLD augments farnesol-mediated apoptosis PLD hydrolysis of PC is via a transphosphatidylation reaction using water as the second substrate for the generation of PA. It has been well characterized that primary alcohols can substitute for water resulting in the formation of a phosphatidylalcohol instead of PA [3]. Phosphatidylalcohols are not substrates for PA phosphatases and consequently inhibit the metabolic pathway through the production of a very poorly metabolized intermediate. The affinity of PLD for alcohol is limited to primary alcohols, whereas secon- dary alcohols are not utilized by PLD. Thus, the addi- tion of primary alcohols to cells in culture effectively inhibits PLD-mediated signaling as the generation of phosphatidylalcohol substantially reduces the forma- tion of PA and its subsequent metabolism to DAG [25–27]. Farnesol addition results in apoptosis in Chi- nese hamster ovary cells (CHO-K1) (Fig. 2A) and we observed that the addition of primary, but not secon- dary, alcohols exacerbated farnesol-induced apoptosis (Fig. 2B). Neither primary nor secondary alcohols alone resulted in the appearance of condensed nuclei or positive Annexin V stain above control levels imply- ing that farnesol is augmenting apoptosis when prod- uct formation by PLD is reduced (Fig. 2B). Although not quantitative, we also assessed cleavage of poly- ADP-ribose polymerase (PARP) and caspase 3 subse- quent to the addition of farnesol to ensure that the observed cell death was indeed apoptotic. Farnesol resulted in cleavage of both PARP and caspase 3 (Fig. 2C,D). The addition of isopropanol or propanol did not result in PARP or caspase cleavage unless farnesol was also present, indicating that the cell death observed was apoptotic. Dissecting the role of phospholipids in the PLD signaling pathway We next determined the ability of PC (upstream of the alcohol block) and DAG (downstream of the alcohol block of PLD) to rescue cells from the augmentation of farnesol-induced apoptosis by primary alcohols. In the presence of primary alcohols DAG, but not PC, rescued farnesol-mediated apoptosis (Fig. 3A). Farne- sol uptake, using radiolabeled farnesol as a probe, was not altered by the exogenous addition of these Fig. 1. DAG consumption for the synthesis of PC and PLD medi- ated turnover for the generation of a DAG-signaling pool. DAG util- ization during the synthesis of PC takes place in the nuclear membrane ⁄ endoplasmic reticulum and the Golgi, while PLD-medi- ated turnover of PC for the production of a DAC occurs primarily at the plasma membrane. M. M. Taylor et al. Farnesol-mediated apoptosis FEBS Journal 272 (2005) 5056–5063 ª 2005 FEBS 5057 glycerolipids (data not shown) indicating that it is the glycerolipids themselves that are providing resistance to farnesol. Although a large number of lipids had been tested for protective properties with respect to farnesol- induced apoptosis, the PLD signaling intermediate PA was not among the published candidates. When exogenous PA was added to CHO-K1 cells prior to the addition of farnesol it was found to be protect- ive in a concentration-dependent manner (Fig. 3B). The data imply that PC is metabolized through PLD and PA phosphatase resulting in the generation of a DAG pool that contributes to a cellular proliferation signal, and farnesol inhibits generation of, or signa- ling by, this DAG pool. However, some studies have indicated that (lyso)PA can be dephosphorylated by cell-surface lipid phosphate phosphatases (LPP) making it difficult to establish a role for PA signa- ling in biological processes through the addition of exogenous lipid [28,29]. In addition, it is difficult to compare exogenous lipid uptake, distribution, and metabolism within a cell. Although our lipid rescue data strongly imply that farnesol is affecting signa- ling by the PLD ⁄ PA phosphatase pathway, we per- formed cell transfection experiments to more directly address this possibility. Active GFP-fusions to open reading frames coding for the two isoforms of PLD (PLD1 and PLD2) [9,10,30–32] and the coupled phosphatidic acid phos- phatase (LPP3) [33], along with a catalytically inactive LPP3 point-mutant [34], were transiently transfected into CHO-K1 cells. The cells were then treated with farnesol and the transfectants positive for enzyme overexpression (as detected by the presence of green fluorescence) were analyzed for apoptosis (Fig. 3C). Increasing PLD1 reduced farnesol-induced apoptosis by nearly 30% and PLD2 overexpression reduced farnesol-induced apoptosis by 60%. Overexpression of LPP3, but not its inactive mutant, completely pre- vented farnesol-induced apoptosis. To determine if farnesol was a direct inhibitor of LPP activity, the PLD-coupled PA phosphatase LPP3 was expressed in Baculovirus and assayed for activity using a Triton X-100 mixed micelle assay [35]. Farnesol was a very poor inhibitor of LPP3 activity as the maximal inhibition observed was 16.5% at 100 lm farnesol (data not shown). Evidence for a DAG responsive target The data argue that farnesol is inhibiting a DAG- responsive target required for life, and that the PLD ⁄ PA phosphatase pathway contributes meaning- A B C D Fig. 2. Farnesol-induced apoptosis in cells incubated with primary alcohols and rescue by phospholipids. CHO-K1 cells were incubated for 4 h with 80 l M farnesol (delivered in dimethylsulfoxide at a final concentration of 0.1% into DMEM supplemented with FBS). (A) Cells were then stained with Ho ¨ echst 33258 or Annexin V and imaged as described in Experimental procedures. (B) The effect of increasing concentrations of isopropanol or propanol on farnesol- induced apoptosis. Apoptosis was determined by imaging at least three random fields of 300 cells in triplicate for both Ho ¨ echst 33258 and Annexin V apoptosis-positive signals. Data are the mean ± SD of at least three separate experiments. (C) Farnesol-induced clea- vage of PARP from its parental form to the caspase-cleaved form. Farnesol was delivered at 80 l M and alcohols were present at 1% (v ⁄ v). (D) Farnesol-induced generation of the caspase-cleaved form of caspase 3. Farnesol was delivered at 80 l M and alcohols were present at 1% (v ⁄ v). Farnesol-mediated apoptosis M. M. Taylor et al. 5058 FEBS Journal 272 (2005) 5056–5063 ª 2005 FEBS fully to this DAG pool. Phorbol esters are non- metabolizable DAG mimics that bind to proteins containing specific C1 domains and activate a similar set of proteins as DAG [4,36]. To test if farnesol- mediated apoptosis required further metabolism of DAG, or if the target of the pathway was a DAG binding protein, we added phorbol ester to cells in the presence or absence of farnesol. The C1 domain binding phorbol ester, b-TPA, inhibited farnesol- mediated apoptosis while its inactive isomer a-TPA did not (Fig. 4). Neither phorbol ester alone altered apoptosis. Discussion Farnesol is a catabolite of the cholesterol ⁄ isoprenoid biosynthetic pathway whose administration preferen- tially induces apoptosis in transformed vs. untrans- formed cells or in tissues taken from cancer patients as Fig. 4. Effect of phorbol esters on farnesol-induced apoptosis. CHO-K1 cells incubated with DMEM for 60 min were pretreated with 100 n M a-TPA or b -TPA for 30 min and then incubated for 2 h with or without 40 l M farnesol (into DMEM delivered in 0.1% dimethylsulfoxide). Apoptosis was determined by imaging at least three random fields of 300 cells in triplicate for both Ho ¨ echst 33258 and Annexin V apoptosis positive signals. The mean ± SEM of four separate experiments is shown. A B C Fig. 3. Role of the PLD pathway in farnesol-induced apoptosis. (A) CHO-K1 cells were preincubated with 65 l M di18 : 1 PC or 65 lM di18 : 1 DAG for 30 min. The cells were then incubated under the same conditions for 4 h with 0, 50, 100 or 150 m M propanol and with 80 l M farnesol (delivered in 0.1% dimethylsulfoxide). Apopto- sis was determined by imaging at least three random fields of 300 cells in triplicate for both Ho ¨ echst 33258 and Annexin V apoptosis positive signals. The mean ± SD of at least three separate experi- ments is shown. Student’s two-tailed t-test was used to determine significant differences (*P < 0.05 from control). (B) CHO-K1 med- ium was replaced by DMEM 60 min prior to the experiment and cells were preincubated with increasing concentrations of phos- phatidic acid added for 15 min. The cells were then incubated under the same conditions for 4 h with 80 l M farnesol (delivered in dimethylsulfoxide at a final concentration of 0.1% into DMEM sup- plemented with FBS). The cells were stained with Ho ¨ echst 33258 or Annexin V to assess apoptosis. The mean ± SD of at least four separate experiments is shown. Student’s two-tailed t-test was used to determine significant differences (*P < 0.05 from no phos- phatidic acid addition). (C) CHO-K1 cells were transfected with vectors containing GFP-tagged constructs of LPP3, a catalytically inactive LPP3 mutant, PLD1, and PLD2. The parent vector, pEGFP (GFP), was used as a control. Growth medium was replaced with DMEM 30 min prior to addition of farnesol at a final concentration of 40 l M (in 0.1% dimethylsulfoxide) for 2 h. The cells were then stained with Ho ¨ echst 33258 or Annexin V and apoptosis was quantitated. Only cells positive for transfection were analyzed. The mean ± SEM of three separate experiments is shown. M. M. Taylor et al. Farnesol-mediated apoptosis FEBS Journal 272 (2005) 5056–5063 ª 2005 FEBS 5059 opposed to healthy control subjects [17,19,37]. We observed that inhibition of PLD signaling augmented farnesol-induced apoptosis, whereas secondary alcohols had no effect on the level of farnesol-induced apopto- sis. Consistent with the importance of DAG signaling is the requirement for PLD conversion of PC to DAG for effective inhibition of farnesol-induced apoptosis. PA, the intermediate in the PLD signaling pathway, was also capable of preventing farnesol-induced apop- tosis. Our evidence implicated PLD signaling as a key mediator of farnesol-induced apoptosis. Overexpression of the PA phosphatase LPP3 pre- vented farnesol-mediated apoptosis. A catalytically inactive LPP3 point mutant was unable to do so, essentially ruling out protein–protein interactions or physical properties of protein overexpression as the cause of rescue. By comparison, expression of PLD1 or PLD2 resulted in a small decrease in farnesol- induced apoptosis [8,10,30,34]. Because LPP3 was the only enzyme in the PLD signaling pathway capable of substantial inhibition of farnesol-induced apoptosis by overexpression, and as we demonstrated that this was not by direct inhibition of LPP3 enzymatic activity by farnesol, this likely means that: (a) LPP3 is the rate- limiting enzyme for production of the farnesol-access- ible DAG pool, and (b) increased flux through this pathway is able to protect cells from farnesol-induced apoptosis by shifting the balance of life-and-death sig- nals away from apoptosis. Our results predict that farnesol is inhibiting a PLD ⁄ LPP3-generated DAG signaling pool. If augmentation of DAG signaling is the key to preventing farnesol-induced apoptosis, it follows that PC rescues farnesol-induced apoptosis only after it has been broken down into a molecule of DAG. Indeed, we observed that primary alcohol aug- mentation of farnesol-mediated apoptosis could be pre- vented by the addition of DAG but not PC. Consistent with this interpretation was the observa- tion that the nonmetabolizable DAG mimetic b-TPA, a pharmacological agent widely used to activate DAG- responsive PKC enzymes and other proteins containing DAG-binding C1 domains [4,36,38,39], also attenuated farnesol-induced apoptosis. As DAG and b-TPA inhib- ited farnesol-induced apoptosis this implies that farnesol prevented direct activation of an antiapoptotic DAG binding target. The nature of this target has yet to be uncovered but it is likely a C1-domain-containing pro- tein that is antiapoptotic and relatively ubiquitous. The C1-domain-containing PKC family member that best fits these criteria is PKCa as it has been generally observed that over-expression of PKCa prevents or attenuates apoptosis in many cell types, whereas down- regulation of PKCa potentiates apoptosis [40–42]. Although the precise role of PKCa? in the regulation of apoptotis is not completely defined it does phosphory- late Bcl-2 on Ser70, an event required for Bcl-2 to inhibit apoptosis [43,44]. Although PKCs are the most thor- oughly characterized phorbol ester receptors in cells, several other C1 domain containing proteins can also bind phorbol esters including the Rac GTPase activating protein n-chimaerin, the scaffolding protein Munc13, some DAG kinase isoforms, and Ras guanine exchange factors (RasGEFs) [45]. Most notable among these are the RasGEFs, as the C1 domain of these proteins is required for their transforming potential and knockout mice display defective proliferative responses [46–48]. The combined data indicate that the PLD ⁄ LPP3 pathway substantially contributes to the generation of an antiapoptotic ⁄ pro-proliferative DAG pool, and signaling by this DAG pool is inhibited by farnesol. The precise farnesol target remains to be determined but our evidence supports a mechanism by which farnesol inhibits DAG activation of an antiapoptotic C1-domain containing protein. Experimental procedures Cell culture and transfection The CHO-K1 cell line was obtained from the American Type Culture Collection. CHO-K1 cells were maintained in a 5% CO 2 atmosphere at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 300 lm proline. CHO-K1 cells were transiently transfected using Lipofectamine (Life Technologies, Rock- ville, MD) at a density of 2 · 10 5 cells in a 60 mm dish. They were grown under normal conditions for a day. Plasmid DNA (2 lg) was added to DMEM and diluted with Lipofec- tamine and the mixture was incubated for 45 min at room temperature. The growth medium was removed from the cells, they were rinsed with DMEM, and the Lipofectamine mixture was overlain on top of the cells. Cells were incubated for 6 h at 37 °C, 5% CO 2 , the transfection solution was removed, and medium containing 10% FBS and 33 lgÆmL )1 proline in DMEM was added to the dishes. Cells were incu- bated at 37 °C, 5% CO 2 for 24 h and the medium was replaced with DMEM supplemented with 5% FBS and 300 lm proline. Cells were routinely cultured for another 24 h before analysis. Lipids were sonicated in 0.1% Tri- ton X-100 and delivered to cells at a concentration not exceeding 0.001% Triton X-100 final concentration. Apoptosis determinations Nuclear morphological changes were monitored using the nuclear stain Ho ¨ echst 33258. Cells were grown on a cover- slip and apoptosis was induced by the desired method. Farnesol-mediated apoptosis M. M. Taylor et al. 5060 FEBS Journal 272 (2005) 5056–5063 ª 2005 FEBS Dishes were placed on ice and the medium was aspirated. Cells were incubated in 4% (v ⁄ v) formaldehyde in NaCl ⁄ P i for 15 min at room temperature. The cells were rinsed twice with freshly prepared ice-cold 5 mm ammonium chloride in NaCl ⁄ P i . The dishes were then incubated for 10 min at 20 °C in 0.05% (w ⁄ v) Triton X-100. The dishes were rinsed twice with NaCl ⁄ P i and then incubated for 10 min at 4 °C in 1% (w ⁄ v) Ho ¨ echst 33258 in NaCl ⁄ P i in the dark. The stain was then rinsed with NaCl ⁄ P i followed by H 2 O. The coverslips were mounted and visualized with a fluorscence microscope (Zeiss Axiovert 200) using an excitation at 365 nm and detection at 480 nm. Dense nuclei (apoptotic) were easily distinguishable from control. The externalization of phosphatidylserine was monitored by annexin V-fluorescein staining using the Annexin-V- FLUOS staining kit from Roche Molecular Biochemicals (Indianapolis, IN) and visualized by fluorescence microscopy as described with propidium iodide was used as a counter- stain for nuclear DNA [24]. At least 300 cells from three random fields were determined for both nuclear DNA mor- phology and annexin-V ⁄ propidium iodide staining from at least three separate experiments. Western blots CHO-K1 cells were seeded at 5 · 10 5 cells per 60 mm dish and grown to 80% confluency. A subset of dishes was trea- ted with 1% isopropanol or 1% propanol for 15 min at 37 °C and 5% CO 2 prior to the addition of 80 lm farnesol in 0.1% dimethylsulfoxide, with an equal volume of dimethylsulfoxide added to control dishes. Cells were incu- bated at 37 °Cin5%CO 2 for 4 h, rinsed twice with cold Tris-buffered saline (TBS), pH 7.5, and then 1 mL of 1% Triton X-100 (v ⁄ v) and CompleteO ˆ protease inhibitor cock- tail (Roche) in TBS was added. Cells were incubated on ice for 10 min and then scraped into microfuge tubes. The tubes were spun at 13 000 g for 10 min in a microfuge. Aliquots were saved for protein assay and the remaining supernatant was precipitated using a final volume of 15% cold acetone overnight at )20 °C. The acetone precipitate was spun at 800 g in a Beckman GS-6 tabletop centrifuge for 5 min. Protein was resuspended in SDS sample buffer to a final concentration of 20 lgÆlL )1 and resolved on a 10% or 15% SDS ⁄ PAGE and transferred to polyvinylidine difluoride membrane (Millipore Corp., Bedford, MA). To detect PARP the membrane was incubated with anti-PARP (Affinity BioReagents, Golden, CO, USA 1 : 1000, v ⁄ v) in 10 mL 5% skim milk-TTBS (skim milk, 5% w ⁄ v; 10 mL TBS, pH 7.5; 4 lL Tween-20) overnight at 4 °C. The blot was rinsed in TBS and incubated with HRP-coupled secon- dary goat anti-(mouse epitope) serum (1 : 5000, v ⁄ v) in 10 mL 5% skim milk TTBS for 1 h. To detect caspase 3 the blots were incubated with anticaspase 3 (Stressgen, Col- legeville, PA, 1 : 1000, v ⁄ v) in 10 mL 5% skim milk–TTBS overnight at 4 °C. Blots were rinsed in TBS and incubated with HRP-coupled secondary goat anti-(rabbit epitope) serum (1 : 5000, v ⁄ v) in 10 mL 5% skim milk–TTBS for 1 h. Actin was probed as a loading control. Blots were incubated with antiactin (Oncogene Research Products, 1 : 5000, v ⁄ v) in 10 mL 5% skim milk-TTBS for 2 h. After rinsing in TBS blots were incubated with HRP-coupled sec- ondary goat anti-(mouse epitope) serum (1 : 5000, v ⁄ v) in 10 mL 5% skim milk–TTBS for 1 h. Proteins were detected using enhanced chemiluminescence following the manufac- turer’s (Amersham Pharmacia Biotech, Piscataway, NJ) instructions. Insect cell culture and phosphatidic acid phosphatase enzyme assay Expression of the PA phosphatase LPP3 in Sf9 cells and determination of LPP activity were exactly as described previously [35]. LPP3 activity was 500–1000-fold over background. 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