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Lithium facilitates apoptotic signaling induced by activation of the Fas death domain-containing receptor

Lithium facilitates apoptotic signaling induced by activation of the Fas death domain-containing... Background: Lithium, a mood stabilizer widely used to treat bipolar disorder, also is a neuroprotectant, providing neurons protection from apoptosis induced by a broad spectrum of toxic conditions. A portion of this neuroprotection is due to lithium's inhibition of glycogen synthase kinase-3. The present investigation examined if the neuroprotection provided by lithium included apoptosis induced by stimulation of the death domain-containing receptor Fas. Results: Instead of providing protection, treatment with 20 mM lithium significantly increased apoptotic signaling induced by activation of Fas, and this occurred in both Jurkat cells and differentiated immortalized hippocampal neurons. Other inhibitors of glycogen synthase kinase-3, including 20 µM indirubin-3'-monoxime, 5 µM kenpaullone, and 5 µM rottlerin, also facilitated Fas- induced apoptotic signaling, indicating that the facilitation of apoptosis by lithium was due to inhibition of glycogen synthase kinase-3. Conclusions: These results demonstrate that lithium is not always a neuroprotectant, and it has the opposite effect of facilitating apoptosis mediated by stimulation of death domain-containing receptors. In addition to stabilizing mood, lithium is a broadly act- Background Lithium has long been the mainstay treatment for bipolar ing cellular protectant, providing neurons and other cells disorder. However, its therapeutic mechanism of action protection from many insults (reviewed in [4-6]). These remains unclear, in part because of the large number of include, but are not limited to, growth factor withdrawal biochemical effects attributed to lithium [1]. Nonetheless, and inhibition of the phosphoinositide 3-kinase (PI3K)/ two actions are prime candidates as lithium's therapeutic Akt signaling pathway [7], treatment with amyloid β-pep- targets, inhibition of inositol monophosphatase [2] and tide [8-11], DNA damage [12], endoplasmic reticulum inhibition of glycogen synthase kinase-3 (GSK3) [3]. Both stress [13], ischemia [14,15], and a variety of toxic agents enzymes are directly inhibited by lithium, but since lith- [5,16,17]. While the mechanistic basis for protection by ium has numerous diverse effects, it is presently unknown lithium in all conditions is not known, in some instances which actions contribute to its therapeutic effects. protection is due to its inhibition of GSK3 [12,13,18-20]. This neuroprotective effect of lithium due to inhibition of GSK3 complements accumulating evidence that GSK3 Page 1 of 7 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 promotes apoptosis in a large number of conditions many other modes of cell death, lithium is not protective (reviewed in [4]). Regardless of the mechanism, the broad following Fas activation, but conversely promotes neuroprotective capacity of lithium has led many investi- apoptosis. gators to suggest the possibility that the therapeutic use of lithium be expanded from mood disorders to also include Results neurodegenerative conditions where lithium may be able Lithium potentiates apoptosis stimulated by Fas in Jurkat cells to retard neuronal dysfunction and death. Jurkat cells were used initially to test if lithium modulates Conspicuously absent from reports of lithium's protective apoptotic signaling induced by activation of Fas. Immu- effects are studies of neuronal apoptosis induced by acti- noblots of active caspase-3 and of a poly(ADP-ribose) vation of death domain-containing receptors, such as Fas polymerase (PARP) 85 kDa cleavage product, which is (also called CD95) and the receptor for tumor necrosis generated by caspase-3-mediated proteolysis, provided factor-α (TNFα). These receptors contain an intracellular indicators of activation of apoptotic signaling. Treatment death domain motif that is required for stimulating apop- with an agonistic anti-Fas antibody (5 to 50 ng/ml) tosis, a major function of these receptors that is initiated caused concentration-dependent increases in active cas- through activation of intracellular proteins and proceeds pase-3 (Fig. 1A) and cleaved PARP (Fig. 1B). Since the Ki to caspase-3 activation [21]. Interestingly, several years of lithium's inhibitory effect on GSK3 is approximately 2 ago lithium was reported to promote the cytotoxic actions mM, a concentration of 20 mM lithium was used to of TNFα [22-24], indicating that lithium's influence on achieve 80–90% inhibition as indicated by previously neuronal responses to stimulation of death domain-con- published concentration-response studies [3]. Pretreat- taining receptors may differ from other conditions in ment with 20 mM lithium (30 min) potentiated Fas- which lithium affords neuroprotection. induced caspase-3 activation by 5.8-fold at the lowest con- centration of agonistic Fas antibody. PARP cleavage Therefore, this study examined the effects of lithium on induced by stimulation of Fas also was potentiated by the activation of apoptotic signaling induced by stimula- lithium, with the greatest potentiation evident at the low- tion of the death domain-containing receptor Fas in two est concentration of agonistic Fas antibody. Treatment types of cells, Jurkat cells and immortalized mouse hip- with lithium alone caused no activation of caspase-3 or pocampal neurons that were differentiated to a neuronal PARP cleavage. Thus, lithium treatment facilitated Fas- phenotype. In both cell types, 20 mM lithium signifi- mediated activation of apoptotic signaling, having the cantly increased caspase-3 activation following stimula- greatest effects at sub-maximal concentrations of Fas tion of Fas. These results demonstrate that in contrast to antibody. Fas-ab(ng/ml) 0 5 5 10 10 20 20 50 50 0 LiCl - - + - + - + - + + 19kDa active caspase-3 17kDa 5.8 4.6 4.3 2.4 Li no Li ±1.8 ±0.9 ±1.1 ±0.4 Fas-ab (ng/ml) 0 5 5 10 10 20 20 50 50 0 LiCl - - + - + - + - + + 89kDa cleaved PARP 27.0 9.0 3.8 1.9 Li no Li ±13.9 ±5.5 ±0.8 ±0.2 Lith Figure 1 ium promotes apoptotic signaling mediated by Fas in Jurkat cells Lithium promotes apoptotic signaling mediated by Fas in Jurkat cells. Jurkat cells were pretreated with 20 mM lithium for 30 min as indicated, followed by treatment with an agonistic anti-Fas antibody (5, 10, 20, or 50 ng/ml). After 24 hr, immunoblots were used to detect (A) active caspase-3, and (B) cleaved PARP. Densitometry was used to measure immunoreactive bands and the ratios of the responses in the presence and absence of lithium were calculated (shown under the immunoblots; Mean ± SEM; n = 3). Page 2 of 7 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 Fas 7h Fas 11h Control Fas 4h M1 M1 M1 M1 4.2 11.0 15.1 15.7 0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 ANNEXIN V FITC ANNEXIN V FITC ANNEXIN V FITC ANNEXIN V FITC LiCl alone LiCl+Fas 4h LiCl+Fas 7h LiCl+Fas 11h M1 34.7 18.4 M1 30.8 M1 7.4 M1 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 ANNEXIN V FITC ANNEXIN V FITC ANNEXIN V FITC ANNEXIN V FITC Lith Figure 2 ium promotes Fas-induced apoptosis in Jurkat cells Lithium promotes Fas-induced apoptosis in Jurkat cells. Flow cytometry with propidium iodide was used to identify apoptotic cells 4, 7, and 11 hr after treatment with anti-Fas (20 ng/ml) in the absence (top row) or presence (bottom row) of 20 mM lithium. Flow cytometry was used to quantitate the time-depend- Treatment of differentiated immortalized hippocampal ence of Jurkat cell death caused by Fas stimulation in the neurons with an agonistic anti-Fas antibody (1 µg/ml), in absence and presence of lithium (Fig. 2). Stimulation of the absence or presence of 20 mM lithium caused a time- Fas caused a time-dependent increase in the number of dependent activation of caspase-3 (Fig. 3A) and of PARP apoptotic cells, and lithium treatment approximately proteolysis (Fig. 3B). Both of these apoptotic responses to doubled Fas-induced apoptosis at all times measured. stimulation of Fas were increased by treatment with 20 Thus, lithium promoted apoptosis induced by stimula- mM lithium (Fig. 3), whereas lithium alone had no effect tion of Fas death domain-containing receptors in Jurkat on these parameters. As indicated by the values given cells. below the western blots, lithium treatment increased Fas- induced caspase-3 activation by approximately two-fold Lithium promotes Fas signaling in hippocampal neurons throughout the experimental time course. The next goal was to identify a neuronal model system in Inhibition of GSK3 facilitates Fas-induced apoptosis which Fas-stimulated apoptosis could be investigated, because few cultured neuronal cell lines express the activation appropriate receptors and signaling activities. Preliminary The two predominantly studied actions of lithium are experiments showed that differentiated immortalized inositol depletion and inhibition of GSK3. Therefore, we hippocampal neurons responded to Fas stimulation with examined if either of these two actions could account for caspase-3 activation and cell death, therefore these cells lithium's facilitation of Fas-induced apoptosis. Inhibition were used to test if lithium modulated this response. of inositol monophosphatase by lithium could conceiva- bly result in depletion of inositol [25] which might facili- tate Fas-induced apoptosis. To test this, cells were Page 3 of 7 (page number not for citation purposes) Events Events Events Events 064 064 Events Events 064 064 Events Events 064 064 BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 Fas-ab (hr) 0 2 2 4 4 8 8 24 24 LiCl - - + - + - + - + active caspase-3 1.4 1.8 2.5 2.1 Li no Li ±0.3 ±0.5 ±0.4 ±0.2 Fas-ab (hr) 0 2 2 4 4 8 8 24 24 LiCl - - + - + - + - + cleaved PARP 2.6 4.7 2.4 3.7 Li no Li ±0.7 ±0.7 ±0.3 ±0.9 Figure 3 Lithium promotes apoptotic signaling mediated by Fas in differentiated immortalized hippocampal neurons Lithium promotes apoptotic signaling mediated by Fas in differentiated immortalized hippocampal neurons. Differentiated immortalized hippocampal neurons were pretreated with 20 mM lithium for 30 min as indicated, followed by treatment with an agonistic anti-Fas antibody (10 µg/ml). Immunoblots were used to measure the time-dependent (A) activation of caspase-3, and (B) PARP cleavage. Ratios of the responses in the presence and absence of lithium are shown under the immunoblots (Mean ± SEM; n = 3–5). pretreated with 20 mM myo-inositol to eliminate any Lithium facilitated apoptotic signaling induced by stimu- potential inositol depletion. This treatment had no effect lation of Fas, and this facilitation by lithium occurred in on Fas-induced apoptotic signaling in the presence or two dissimilar types of cells, Jurkat cells and differentiated absence of lithium (Fig. 4A), indicating that inositol hippocampal neurons. These findings extend to Fas, and depletion did not account for the facilitation of caspase-3 to neurons, previous reports that lithium promotes TNFα- activation caused by lithium. induced cytotoxicity [22-24,29]. Lithium and another spe- cific inhibitor of GSK3 also recently were reported to To test if Fas-induced apoptosis was facilitated by lith- enhance apoptosis induced by tumor necrosis factor- ium's inhibition of GSK3, additional GSK3 inhibitors related apoptosis-inducing ligand (TRAIL) in human were tested, including 20 µM indirubin-3'-monoxime prostate cancer cell lines [30]. Taken together, it is evident [26], 5 µM kenpaullone [27], and 5 µM rottlerin [28]. As that apoptosis induced by activation of death domain- with lithium, to varying degrees each of these GSK3 inhib- containing receptors is facilitated by lithium, as opposed itors also increased Fas-induced caspase-3 activation and to the protective action of lithium in many other condi- PARP proteolysis in both Jurkat cells and differentiated tions. For example, pretreatment with a high concentra- hippocampal cells (Fig. 4B). These findings indicate that tion of lithium (10 to 30 mM) previously was reported to inhibition of GSK3 facilitates Fas-induced caspase protect cells from apoptosis and/or loss of viability caused activation. by amyloid β-peptide [8,11], withdrawal of nerve growth factor [31], DNA damage [12], hypoxia [32], trophic fac- Discussion tor withdrawal [33,34], hypertonic stress [35], potassium- The results of this study demonstrate for the first time that withdrawal or inhibition of PI3K [36], endoplasmic retic- lithium and other GSK3 inhibitors promote death ulum stress [13], platelet activating factor [37], rotenone, domain-containing receptor-mediated apoptosis in neu- and 1-methyl-4-phenylpyridinium (MPP) [17]. This dif- ral cells, and that Fas-mediated apoptotic signaling is ferent effect of lithium among apoptotic conditions is facilitated by lithium. Thus, in contrast to much current likely related to the mechanisms mediating the two major literature, lithium is not always neuroprotective and GSK3 classes of apoptosis: intrinsic and extrinsic apoptosis [38]. is not always pro-apoptotic. Many, if not all, of the conditions in which lithium is pro- tective appear to activate the intrinsic apoptotic signaling Page 4 of 7 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 active caspase-3 cleaved PARP Ctl - Li Li+inos inos inos Li Li+inos + Fas-ab B Jurkat cells Hippocampal cells active caspase-3 cleaved PARP Ctl - Li Rott Ind Ken Ctl - Li Ind Rott Ken + Fas-ab + Fas-ab GSK3 inhibit Figure 4 ors facilitate Fas-induced apoptosis GSK3 inhibitors facilitate Fas-induced apoptosis. (A) Differentiated immortalized hippocampal neurons were pretreated for 30 min with 20 mM lithium, with or without 20 mM myo-inositol, and active caspase-3 and proteolyzed PARP were measured 24 hr with or without Fas stimulation. (B) In Jurkat cells and differentiated immortalized hippocampal neurons, activation of cas- pase-3 and PARP cleavage induced by Fas stimulation were facilitated following 30 min pretreatment with 20 mM lithium, 5 µM rottlerin (Rott), 20 µM indirubin-3'-monoxime (Ind), or 5 µM kenpaullone (Ken). pathway. In contrast, the extrinsic apoptosis pathway is These results indicate that GSK3 attenuates extrinsic apop- induced by activation of Fas, TRAIL, and TNFα receptors. tosis, and that lithium and other GSK3 inhibitors block The fundamental differences in these apoptotic mecha- this effect to promote extrinsic apoptosis. As a corollary, nisms appear linked to the differential effects of lithium. this raises the question of whether or not such an action of lithium could occur in vivo in humans treated with The mechanism by which lithium promotes the cytotoxic- therapeutic concentrations of lithium. Initially, when lith- ity caused by stimulation of Fas appears due to inhibition ium's direct inhibition of GSK3 was described [3] investi- of GSK3 because other GSK3 inhibitors had the same gators were skeptical that this could have any therapeutic effect. This is in accordance with the conclusions that lith- relevance because the Ki for inhibition is about 2 mM, ium's inhibition of GSK3 facilitates apoptosis induced by much above the therapeutic level of near 1 mM. However, TRAIL [30] and by TNFα [39], and that TNFα-induced lithium has the intriguing ability to inhibit GSK3 in two hepatotoxicity was potentiated by elimination of GSK3β ways in vivo. First it causes direct inhibition. Second, this or treatment with lithium [40]. However, other investiga- direct inhibition at a low concentration is amplified in tors concluded that lithium's facilitation of TNFα-induced vivo after chronic lithium treatment by an increase in the apoptosis was independent of GSK3 inhibition [29]. In inhibitory serine-phosphorylation of GSK3 [41]. These contrast with our findings, Schotte et al [29] reported that dual mechanisms were recently reviewed [6]. The mecha- in stable lines of fibrosarcoma cells transfected with Fas nism for this dual inhibition was recently proposed by there was no potentiation by lithium of agonistic anti-Fas- Klein's group to be due to regulation of phosphatases act- induced cell death. Whether this difference from our ing on GSK3 [42]. Therefore, although high lithium con- results is caused by different cell types, over-expressed Fas, centrations are necessary to cause substantial inhibition of or other reasons is unknown. We speculate that since we GSK3 in acute in vitro experiments, the effects of lower observed the greatest effects of lithium at sub-maximal lithium levels achieved in vivo after chronic administra- activation of Fas, facilitation by lithium might be difficult tion are amplified by this mechanism. If this amplifica- to detect in cells overexpressing Fas. tion mechanism contributes to lithium's inhibition of GSK3 in vivo, and inhibition of GSK3 occurs to a signifi- Page 5 of 7 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 cant extent in vivo with a therapeutically relevant concen- Authors' contributions tration of lithium, then the same rationale suggests that LS carried out all of the experiments. TZ and RSJ con- lithium also may facilitate extrinsic apoptosis in vivo ceived, designed, and analyzed the experiments. All dependent on the magnitude of inhibition of GSK3 that is authors read and approved the final manuscript. necessary for this effect. Acknowledgements This research was supported by grants from the National Institutes of Conclusions Health. These experiments demonstrated that lithium facilitates Fas-induced apoptotic signaling in Jurkat cells and in dif- References ferentiated hippocampal neurons. Along with previous 1. Jope RS: Anti-bipolar therapy: mechanism of action of reports that lithium potentiates apoptosis induced by lithium. Mol Psychiatry 1999, 4:117-128. TNFα or TRAIL, it may be possible to generalize that lith- 2. Hallcher LM, Sherman WR: The effects of lithium ion and other agents on the activity of myo-inositol-1-phosphatase from ium facilitates extrinsic apoptotic signaling by death bovine brain. J Biol Chem 1980, 255:10896-10901. domain-containing receptors, as opposed to its protective 3. 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Lithium facilitates apoptotic signaling induced by activation of the Fas death domain-containing receptor

Song, Ling; Zhou, Tong; Jope, Richard
BMC Neuroscience , Volume 5 (1) – May 24, 2004
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Copyright © 2004 by Song et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurobiology; Animal Models
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Abstract

Background: Lithium, a mood stabilizer widely used to treat bipolar disorder, also is a neuroprotectant, providing neurons protection from apoptosis induced by a broad spectrum of toxic conditions. A portion of this neuroprotection is due to lithium's inhibition of glycogen synthase kinase-3. The present investigation examined if the neuroprotection provided by lithium included apoptosis induced by stimulation of the death domain-containing receptor Fas. Results: Instead of providing protection, treatment with 20 mM lithium significantly increased apoptotic signaling induced by activation of Fas, and this occurred in both Jurkat cells and differentiated immortalized hippocampal neurons. Other inhibitors of glycogen synthase kinase-3, including 20 µM indirubin-3'-monoxime, 5 µM kenpaullone, and 5 µM rottlerin, also facilitated Fas- induced apoptotic signaling, indicating that the facilitation of apoptosis by lithium was due to inhibition of glycogen synthase kinase-3. Conclusions: These results demonstrate that lithium is not always a neuroprotectant, and it has the opposite effect of facilitating apoptosis mediated by stimulation of death domain-containing receptors. In addition to stabilizing mood, lithium is a broadly act- Background Lithium has long been the mainstay treatment for bipolar ing cellular protectant, providing neurons and other cells disorder. However, its therapeutic mechanism of action protection from many insults (reviewed in [4-6]). These remains unclear, in part because of the large number of include, but are not limited to, growth factor withdrawal biochemical effects attributed to lithium [1]. Nonetheless, and inhibition of the phosphoinositide 3-kinase (PI3K)/ two actions are prime candidates as lithium's therapeutic Akt signaling pathway [7], treatment with amyloid β-pep- targets, inhibition of inositol monophosphatase [2] and tide [8-11], DNA damage [12], endoplasmic reticulum inhibition of glycogen synthase kinase-3 (GSK3) [3]. Both stress [13], ischemia [14,15], and a variety of toxic agents enzymes are directly inhibited by lithium, but since lith- [5,16,17]. While the mechanistic basis for protection by ium has numerous diverse effects, it is presently unknown lithium in all conditions is not known, in some instances which actions contribute to its therapeutic effects. protection is due to its inhibition of GSK3 [12,13,18-20]. This neuroprotective effect of lithium due to inhibition of GSK3 complements accumulating evidence that GSK3 Page 1 of 7 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 promotes apoptosis in a large number of conditions many other modes of cell death, lithium is not protective (reviewed in [4]). Regardless of the mechanism, the broad following Fas activation, but conversely promotes neuroprotective capacity of lithium has led many investi- apoptosis. gators to suggest the possibility that the therapeutic use of lithium be expanded from mood disorders to also include Results neurodegenerative conditions where lithium may be able Lithium potentiates apoptosis stimulated by Fas in Jurkat cells to retard neuronal dysfunction and death. Jurkat cells were used initially to test if lithium modulates Conspicuously absent from reports of lithium's protective apoptotic signaling induced by activation of Fas. Immu- effects are studies of neuronal apoptosis induced by acti- noblots of active caspase-3 and of a poly(ADP-ribose) vation of death domain-containing receptors, such as Fas polymerase (PARP) 85 kDa cleavage product, which is (also called CD95) and the receptor for tumor necrosis generated by caspase-3-mediated proteolysis, provided factor-α (TNFα). These receptors contain an intracellular indicators of activation of apoptotic signaling. Treatment death domain motif that is required for stimulating apop- with an agonistic anti-Fas antibody (5 to 50 ng/ml) tosis, a major function of these receptors that is initiated caused concentration-dependent increases in active cas- through activation of intracellular proteins and proceeds pase-3 (Fig. 1A) and cleaved PARP (Fig. 1B). Since the Ki to caspase-3 activation [21]. Interestingly, several years of lithium's inhibitory effect on GSK3 is approximately 2 ago lithium was reported to promote the cytotoxic actions mM, a concentration of 20 mM lithium was used to of TNFα [22-24], indicating that lithium's influence on achieve 80–90% inhibition as indicated by previously neuronal responses to stimulation of death domain-con- published concentration-response studies [3]. Pretreat- taining receptors may differ from other conditions in ment with 20 mM lithium (30 min) potentiated Fas- which lithium affords neuroprotection. induced caspase-3 activation by 5.8-fold at the lowest con- centration of agonistic Fas antibody. PARP cleavage Therefore, this study examined the effects of lithium on induced by stimulation of Fas also was potentiated by the activation of apoptotic signaling induced by stimula- lithium, with the greatest potentiation evident at the low- tion of the death domain-containing receptor Fas in two est concentration of agonistic Fas antibody. Treatment types of cells, Jurkat cells and immortalized mouse hip- with lithium alone caused no activation of caspase-3 or pocampal neurons that were differentiated to a neuronal PARP cleavage. Thus, lithium treatment facilitated Fas- phenotype. In both cell types, 20 mM lithium signifi- mediated activation of apoptotic signaling, having the cantly increased caspase-3 activation following stimula- greatest effects at sub-maximal concentrations of Fas tion of Fas. These results demonstrate that in contrast to antibody. Fas-ab(ng/ml) 0 5 5 10 10 20 20 50 50 0 LiCl - - + - + - + - + + 19kDa active caspase-3 17kDa 5.8 4.6 4.3 2.4 Li no Li ±1.8 ±0.9 ±1.1 ±0.4 Fas-ab (ng/ml) 0 5 5 10 10 20 20 50 50 0 LiCl - - + - + - + - + + 89kDa cleaved PARP 27.0 9.0 3.8 1.9 Li no Li ±13.9 ±5.5 ±0.8 ±0.2 Lith Figure 1 ium promotes apoptotic signaling mediated by Fas in Jurkat cells Lithium promotes apoptotic signaling mediated by Fas in Jurkat cells. Jurkat cells were pretreated with 20 mM lithium for 30 min as indicated, followed by treatment with an agonistic anti-Fas antibody (5, 10, 20, or 50 ng/ml). After 24 hr, immunoblots were used to detect (A) active caspase-3, and (B) cleaved PARP. Densitometry was used to measure immunoreactive bands and the ratios of the responses in the presence and absence of lithium were calculated (shown under the immunoblots; Mean ± SEM; n = 3). Page 2 of 7 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 Fas 7h Fas 11h Control Fas 4h M1 M1 M1 M1 4.2 11.0 15.1 15.7 0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 ANNEXIN V FITC ANNEXIN V FITC ANNEXIN V FITC ANNEXIN V FITC LiCl alone LiCl+Fas 4h LiCl+Fas 7h LiCl+Fas 11h M1 34.7 18.4 M1 30.8 M1 7.4 M1 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 ANNEXIN V FITC ANNEXIN V FITC ANNEXIN V FITC ANNEXIN V FITC Lith Figure 2 ium promotes Fas-induced apoptosis in Jurkat cells Lithium promotes Fas-induced apoptosis in Jurkat cells. Flow cytometry with propidium iodide was used to identify apoptotic cells 4, 7, and 11 hr after treatment with anti-Fas (20 ng/ml) in the absence (top row) or presence (bottom row) of 20 mM lithium. Flow cytometry was used to quantitate the time-depend- Treatment of differentiated immortalized hippocampal ence of Jurkat cell death caused by Fas stimulation in the neurons with an agonistic anti-Fas antibody (1 µg/ml), in absence and presence of lithium (Fig. 2). Stimulation of the absence or presence of 20 mM lithium caused a time- Fas caused a time-dependent increase in the number of dependent activation of caspase-3 (Fig. 3A) and of PARP apoptotic cells, and lithium treatment approximately proteolysis (Fig. 3B). Both of these apoptotic responses to doubled Fas-induced apoptosis at all times measured. stimulation of Fas were increased by treatment with 20 Thus, lithium promoted apoptosis induced by stimula- mM lithium (Fig. 3), whereas lithium alone had no effect tion of Fas death domain-containing receptors in Jurkat on these parameters. As indicated by the values given cells. below the western blots, lithium treatment increased Fas- induced caspase-3 activation by approximately two-fold Lithium promotes Fas signaling in hippocampal neurons throughout the experimental time course. The next goal was to identify a neuronal model system in Inhibition of GSK3 facilitates Fas-induced apoptosis which Fas-stimulated apoptosis could be investigated, because few cultured neuronal cell lines express the activation appropriate receptors and signaling activities. Preliminary The two predominantly studied actions of lithium are experiments showed that differentiated immortalized inositol depletion and inhibition of GSK3. Therefore, we hippocampal neurons responded to Fas stimulation with examined if either of these two actions could account for caspase-3 activation and cell death, therefore these cells lithium's facilitation of Fas-induced apoptosis. Inhibition were used to test if lithium modulated this response. of inositol monophosphatase by lithium could conceiva- bly result in depletion of inositol [25] which might facili- tate Fas-induced apoptosis. To test this, cells were Page 3 of 7 (page number not for citation purposes) Events Events Events Events 064 064 Events Events 064 064 Events Events 064 064 BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 Fas-ab (hr) 0 2 2 4 4 8 8 24 24 LiCl - - + - + - + - + active caspase-3 1.4 1.8 2.5 2.1 Li no Li ±0.3 ±0.5 ±0.4 ±0.2 Fas-ab (hr) 0 2 2 4 4 8 8 24 24 LiCl - - + - + - + - + cleaved PARP 2.6 4.7 2.4 3.7 Li no Li ±0.7 ±0.7 ±0.3 ±0.9 Figure 3 Lithium promotes apoptotic signaling mediated by Fas in differentiated immortalized hippocampal neurons Lithium promotes apoptotic signaling mediated by Fas in differentiated immortalized hippocampal neurons. Differentiated immortalized hippocampal neurons were pretreated with 20 mM lithium for 30 min as indicated, followed by treatment with an agonistic anti-Fas antibody (10 µg/ml). Immunoblots were used to measure the time-dependent (A) activation of caspase-3, and (B) PARP cleavage. Ratios of the responses in the presence and absence of lithium are shown under the immunoblots (Mean ± SEM; n = 3–5). pretreated with 20 mM myo-inositol to eliminate any Lithium facilitated apoptotic signaling induced by stimu- potential inositol depletion. This treatment had no effect lation of Fas, and this facilitation by lithium occurred in on Fas-induced apoptotic signaling in the presence or two dissimilar types of cells, Jurkat cells and differentiated absence of lithium (Fig. 4A), indicating that inositol hippocampal neurons. These findings extend to Fas, and depletion did not account for the facilitation of caspase-3 to neurons, previous reports that lithium promotes TNFα- activation caused by lithium. induced cytotoxicity [22-24,29]. Lithium and another spe- cific inhibitor of GSK3 also recently were reported to To test if Fas-induced apoptosis was facilitated by lith- enhance apoptosis induced by tumor necrosis factor- ium's inhibition of GSK3, additional GSK3 inhibitors related apoptosis-inducing ligand (TRAIL) in human were tested, including 20 µM indirubin-3'-monoxime prostate cancer cell lines [30]. Taken together, it is evident [26], 5 µM kenpaullone [27], and 5 µM rottlerin [28]. As that apoptosis induced by activation of death domain- with lithium, to varying degrees each of these GSK3 inhib- containing receptors is facilitated by lithium, as opposed itors also increased Fas-induced caspase-3 activation and to the protective action of lithium in many other condi- PARP proteolysis in both Jurkat cells and differentiated tions. For example, pretreatment with a high concentra- hippocampal cells (Fig. 4B). These findings indicate that tion of lithium (10 to 30 mM) previously was reported to inhibition of GSK3 facilitates Fas-induced caspase protect cells from apoptosis and/or loss of viability caused activation. by amyloid β-peptide [8,11], withdrawal of nerve growth factor [31], DNA damage [12], hypoxia [32], trophic fac- Discussion tor withdrawal [33,34], hypertonic stress [35], potassium- The results of this study demonstrate for the first time that withdrawal or inhibition of PI3K [36], endoplasmic retic- lithium and other GSK3 inhibitors promote death ulum stress [13], platelet activating factor [37], rotenone, domain-containing receptor-mediated apoptosis in neu- and 1-methyl-4-phenylpyridinium (MPP) [17]. This dif- ral cells, and that Fas-mediated apoptotic signaling is ferent effect of lithium among apoptotic conditions is facilitated by lithium. Thus, in contrast to much current likely related to the mechanisms mediating the two major literature, lithium is not always neuroprotective and GSK3 classes of apoptosis: intrinsic and extrinsic apoptosis [38]. is not always pro-apoptotic. Many, if not all, of the conditions in which lithium is pro- tective appear to activate the intrinsic apoptotic signaling Page 4 of 7 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 active caspase-3 cleaved PARP Ctl - Li Li+inos inos inos Li Li+inos + Fas-ab B Jurkat cells Hippocampal cells active caspase-3 cleaved PARP Ctl - Li Rott Ind Ken Ctl - Li Ind Rott Ken + Fas-ab + Fas-ab GSK3 inhibit Figure 4 ors facilitate Fas-induced apoptosis GSK3 inhibitors facilitate Fas-induced apoptosis. (A) Differentiated immortalized hippocampal neurons were pretreated for 30 min with 20 mM lithium, with or without 20 mM myo-inositol, and active caspase-3 and proteolyzed PARP were measured 24 hr with or without Fas stimulation. (B) In Jurkat cells and differentiated immortalized hippocampal neurons, activation of cas- pase-3 and PARP cleavage induced by Fas stimulation were facilitated following 30 min pretreatment with 20 mM lithium, 5 µM rottlerin (Rott), 20 µM indirubin-3'-monoxime (Ind), or 5 µM kenpaullone (Ken). pathway. In contrast, the extrinsic apoptosis pathway is These results indicate that GSK3 attenuates extrinsic apop- induced by activation of Fas, TRAIL, and TNFα receptors. tosis, and that lithium and other GSK3 inhibitors block The fundamental differences in these apoptotic mecha- this effect to promote extrinsic apoptosis. As a corollary, nisms appear linked to the differential effects of lithium. this raises the question of whether or not such an action of lithium could occur in vivo in humans treated with The mechanism by which lithium promotes the cytotoxic- therapeutic concentrations of lithium. Initially, when lith- ity caused by stimulation of Fas appears due to inhibition ium's direct inhibition of GSK3 was described [3] investi- of GSK3 because other GSK3 inhibitors had the same gators were skeptical that this could have any therapeutic effect. This is in accordance with the conclusions that lith- relevance because the Ki for inhibition is about 2 mM, ium's inhibition of GSK3 facilitates apoptosis induced by much above the therapeutic level of near 1 mM. However, TRAIL [30] and by TNFα [39], and that TNFα-induced lithium has the intriguing ability to inhibit GSK3 in two hepatotoxicity was potentiated by elimination of GSK3β ways in vivo. First it causes direct inhibition. Second, this or treatment with lithium [40]. However, other investiga- direct inhibition at a low concentration is amplified in tors concluded that lithium's facilitation of TNFα-induced vivo after chronic lithium treatment by an increase in the apoptosis was independent of GSK3 inhibition [29]. In inhibitory serine-phosphorylation of GSK3 [41]. These contrast with our findings, Schotte et al [29] reported that dual mechanisms were recently reviewed [6]. The mecha- in stable lines of fibrosarcoma cells transfected with Fas nism for this dual inhibition was recently proposed by there was no potentiation by lithium of agonistic anti-Fas- Klein's group to be due to regulation of phosphatases act- induced cell death. Whether this difference from our ing on GSK3 [42]. Therefore, although high lithium con- results is caused by different cell types, over-expressed Fas, centrations are necessary to cause substantial inhibition of or other reasons is unknown. We speculate that since we GSK3 in acute in vitro experiments, the effects of lower observed the greatest effects of lithium at sub-maximal lithium levels achieved in vivo after chronic administra- activation of Fas, facilitation by lithium might be difficult tion are amplified by this mechanism. If this amplifica- to detect in cells overexpressing Fas. tion mechanism contributes to lithium's inhibition of GSK3 in vivo, and inhibition of GSK3 occurs to a signifi- Page 5 of 7 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/20 cant extent in vivo with a therapeutically relevant concen- Authors' contributions tration of lithium, then the same rationale suggests that LS carried out all of the experiments. TZ and RSJ con- lithium also may facilitate extrinsic apoptosis in vivo ceived, designed, and analyzed the experiments. All dependent on the magnitude of inhibition of GSK3 that is authors read and approved the final manuscript. necessary for this effect. Acknowledgements This research was supported by grants from the National Institutes of Conclusions Health. These experiments demonstrated that lithium facilitates Fas-induced apoptotic signaling in Jurkat cells and in dif- References ferentiated hippocampal neurons. Along with previous 1. Jope RS: Anti-bipolar therapy: mechanism of action of reports that lithium potentiates apoptosis induced by lithium. Mol Psychiatry 1999, 4:117-128. TNFα or TRAIL, it may be possible to generalize that lith- 2. Hallcher LM, Sherman WR: The effects of lithium ion and other agents on the activity of myo-inositol-1-phosphatase from ium facilitates extrinsic apoptotic signaling by death bovine brain. J Biol Chem 1980, 255:10896-10901. domain-containing receptors, as opposed to its protective 3. 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Rao R, Hao CM, Breyer MD: Hypertonic stress activates glyco- Publish with Bio Med Central and every gen synthase kinase 3β-mediated apoptosis of renal medul- scientist can read your work free of charge lary interstitial cells, suppressing an NFκB-driven cyclooxygenase-2-dependent survival pathway. J Biol Chem "BioMed Central will be the most significant development for 2004, 279:3949-3955. disseminating the results of biomedical researc h in our lifetime." 36. Cross DA, Culbert AA, Chalmers KA, Facci L, Skaper SD, Reith AD: Sir Paul Nurse, Cancer Research UK Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Your research papers will be: Neurochem 2001, 77:94-102. available free of charge to the entire biomedical community 37. 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