Glucocorticoid-Induced Apoptosis Revisited

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Mar 14, 2006 - Dahlman-Wright K, Wright A, Gustafsson JA, Carlstedt-Duke J. .... Helmberg A, Auphan N, Caelles C, Karin M. Glucocorticoid-induced ...
[Cell Cycle 5:10, 1017-1026, 15 May 2006]; ©2006 Landes Bioscience

Glucocorticoid-Induced Apoptosis Revisited Perspective

A Novel Role for Glucocorticoid Receptor Translocation to the Mitochondria ABSTRACT Recent data cast new light on the mechanisms by which glucocorticoids (GCs) elicit apoptosis of thymocytes and leukemia cells. Here we attempt to integrate recent studies by others and us, which provide a novel insight to this apoptotic process. In the last few years it was made clear that there is a tight cooperation between genomic and nongenomic effects exerted by GC receptors (GRs). GC invokes major alterations in the gene expression profile through GR-mediated transactivation and transrepression, which ultimately tip the balance between pro-survival and pro-apoptotic proteins. Although essential in shaping the cell’s proteome, these genomic effects are insufficient to elicit apoptotic death and additional signals are required for activating the pro-apoptotic proteins. Several non-genomic effects have been described that occur immediately following exposure to GC, which are imperative for the induction of apoptosis. We have recently observed that GC induces instant GR translocation to the mitochondria in GC-sensitive, but not in GC-resistant, T lymphoid cells. This response contrasts the nuclear translocation of GR occurring in both cell types. We propose that the sustained elevation of GR in the mitochondria following GC exposure is crucial for triggering apoptosis.

*Correspondence to: Eitan Yefenof; The Lautenberg Center for General and Tumor Immunology; The Hebrew University—Hadassah Medical School; P.O.Box 12272; 91120 Jerusalem, Israel; Tel.: +972.2.675.8726; Fax: +972.2.643.0834; Email: [email protected] Original manuscript submitted: 03/14/06 Manuscript accepted: 03/23/06

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Omega Fraternity; Jerusalem, Israel

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2Department of Pharmacology; Faculty of Dental Medicine Founded by the Alpha-

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1The Lautenberg Center for General and Tumor Immunology; The Hebrew University—Hadassah Medical School; Jerusalem, Israel

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Ronit Vogt Sionov1 Shlomit Kfir1 Elazar Zafrir1 Orly Cohen1 Yael Zilberman2 Eitan Yefenof1,*

INTRODUCTION

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2738

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apoptosis, glucocorticoid, glucocorticoid receptor, leukemia, mitochondria, non-genomic effects

Already in the 1940’s glucocorticoids (GCs) were found effective in harnessing the growth of leukemic tumors, and subsequently introduced as the first line drug in the treatment of childhood acute lymphoblastic leukemia (ALL). Later studies indicated that GCs are potent inducers of apoptosis in thymocytes and leukemic cells, which provided the basis for their clinical usefulness. Today GCs constitute central components in the treatment of various hematological malignancies such as ALL, multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma, besides their wide use as anti-inflammatory drugs in autoimmune and inflammatory diseases.1-4 Despite their prevalent clinical application, the mechanisms by which GC induces apoptosis remain obscure. Hence, an insight into the molecular pathways underlying GC-mediated cell death is crucial for enhancing the efficacy of GC therapy on the one hand, while minimizing the adverse effects of the drug on the other hand. Several reviews have been published on this topic during the last couple of years.1,5-9 To avoid redundancy, we shall restrict ourselves in the present article to recent data that provide better understanding of GC-induced apoptosis with specific emphasis on the induction phase. First we will deal with factors affecting the cell’s decision to live or die following GC treatment, and then describe the genomic and non-genomic effects of GC required for initiating the signaling pathways leading to apoptosis.

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acute lymphoblastic leukemia cyclin-dependent kinase chronic lymphocytic leukemia chronic myeloid leukemia DNA-binding domain dexamethasone glucocorticoid glucocorticoid receptor glucocorticoid response element heat shock proteins ligand binding domain mitochondrial localization signal multiple myeloma nuclear export signal nuclear localization signal promyelocytic leukemia reactive oxygen species

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ALL CDK CLL CML DBD Dex GC GR GRE HSP LBD MLS MM NES NLS PML ROS

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ABBREVIATIONS

ACKNOWLEDGEMENTS See page 1023.

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A DECISION BETWEEN LIFE AND DEATH

The requirement for adequate expression of glucocorticoid receptors. Apoptosis by GC requires its binding to the glucocorticoid receptor (GR). Thus far, only one GR encoding gene has been identified, located at chromosome 5 (5q31) in human.10 However, several GR mRNA transcripts are formed due to utilization of different promoters.11,12 In contrast to the 1B-1E transcripts displayed in all tissues analyzed, the 1A transcript shows tissue-specificity and is mainly expressed in thymocytes, T leukemic cells and the brain cortex.11,13 Accordingly, the 1A GR mRNA has been implicated in GC-induced apoptosis.11,13 However, it is unclear why this particular transcript should confer higher GC sensitivity since it is translated to the same GR protein as the 1B-1E Cell Cycle

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Figure1. (A) Basal expression of GR, anti-apoptotic and pro-apoptotic proteins in various leukemia and lymphoma cells. Total lysate of each cell line was analysed by Western blot using (a) PAI-511A antibodies to GR (Affinity Reagents); (b) B46620 antibodies to Bcl-2 (BD Transduction Labs); (c) H-5 antibodies to Bcl-XL (Santa Cruz Biotech.); (d) S-19 antibodies to Mcl-1 (Santa Cruz Biotech.); (e and f) PC66 antibodies to Bax (Oncogene Research Products); (g) AM04 antibodies to Bak (Oncogene Research Products); (h) 202000 antibodies to Bim (Calbiochem); (i) B36420 antibodies to Bad (BD Transduction Labs); (j) Polyclonal antibodies to Bid (kindly provided by Dr. A. Gross, The Weizmann Institute, Rehovot, Israel); (k) E-20 antibodies to NBK (Santa Cruz Biotech.) and (l) DM1A antibodies to α-tubulin (Sigma). It should be noted that the PA1-511A antibodies to GR stain two major bands, the upper one is GRα. The nature of the lower band is uncertain, but it reacts with both the N-terminal M20 antibodies and the C-terminal P20 antibodies to GR,19 thereby excluding GRβ and GR-B. It is not immunoprecipitated by the PA1-511A, FiGR and M20 antibodies, and therefore considered here as a background band. SH: Highly sensitive cells; SL: Low responding cells; R: Resistant cells. (B) The total GR expression level in PD1.6 (a), B10 (c), thymocytes (e) and splenocytes (g) is not altered by Dex. The cells were incubated with various concentrations of Dex for five hours, prior to total extract and Western blotting using PA1-511A antibodies to GR (a and c) or M20 antibodies to GR (Santa Cruz Biotech.; e and g). The blots were reprobed with antibodies to α-tubulin (b, d, f and h). (C) Effect of Dex treatment on the GR expression level in other cell lines. The cells were either untreated or treated with 100nM Dex for two hours, prior to total lysate and Western blotting using PA1-511A antibodies to GR (a) and DM1A antibodies to α-tubulin (b).

transcripts. Although various GR isoforms can be generated due to alternative splicing and different initiation sites,14,15 the pro-apoptotic activity has mainly been attributed to the full-length 94kD GRα, which is the major product of all GR transcripts.13,16-20 Several studies have shown that a threshold level of GRα is mandatory for conferring GC-sensitivity to the cells.5,16-18 When this prerequisite is fulfilled, a quantitative correlation between GR expression and GC-sensitivity is observed in GC-responding lymphoid cells.18,21,22 However, the expression of GR per se is insufficient to turn on an 1018

apoptotic response as GC-resistant cells may express similar and even higher GR levels than GC-sensitive lymphoid cells19,23-25 (Fig. 1A). Although all GR mRNA transcripts are displayed in thymocytes and leukemic cells, only the 1A transcript is upregulated by GC with concomitant increase in the level of GR translated from this transcript.11,13 This effect is regulated by a glucocorticoid response element (GRE)-like sequence located downstream to the 1A transcriptional initiation site.11,13 Across the board increase of total GR expression level in response to GC has been only observed in few leukemic cells

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such as the low responding CCRF-CEM ALL cells,11,26,27 whereas in some others, e.g., the GC-resistant IM-9 MM cells, GC exposure led to a decreased GR expression.11,27 In other cells, e.g., the GC-resistant Jurkat line, GC treatment did not affect the level of GR.26 A question ensuing from these data is whether upregulation of GR expression is a general prerequisite for sensitizing lymphoid cells to GC-induced apoptosis. To address this question, we studied a series of leukemia and lymphoma cells, which can be divided into three major groups according to their susceptibility to GC-induced apoptosis. The first group (I) consisted of highly sensitive cells represented by the prototypic CD4+8+ PD1.6 thymic lymphoma and 2B4 T hybridoma cells (50–90% apoptosis following 20-hrs incubation with 100nM Dex). The second, low responding group (II), included CCRF-CEM ALL, BV173 chronic myeloid leukemia (CML), CD4-8- B10 thymic lymphoma and CD4-8- S49 T cell lymphoma. These cells did not undergo apoptosis even after 24 hrs of incubation with 1µM Dex. However, when treated with 100nM-1µM Dex for 48 hrs, 15–40% of the cells died. The third group (III) represented cells that are totally resistant to GC, including NB4 promyelocytic leukemia (PML), K562 CML and EL-4 T cell lymphoma. The rapid GC-induced cell death of group I shows typical apoptotic features, whereas the delayed death of group II displays characteristics resembling necrosis.9 The basal GR levels vary between the cell lines (Fig. 1A, a), and may even be higher in GC-resistant and low responding cells than in the highly sensitive ones. Thus, additional factors beyond GR expression level dictate the outcome of the GC response. In order to study whether GC-induced GR upregulation is a hallmark of GC-sensitive cells, we analyzed the GR expression level in the highly GC-sensitive PD1.6 cells19 and in the low responding B10 cells19 after incubation with various concentrations of the synthetic GC Dexamethasone (Dex). These two cell lines express similar basal GR levels19 (Fig. 1A, a, lanes 1 and 2). Dex treatment had no effect on the GR expression levels in these cells (Fig. 1B, a and c), thus excluding GR upregulation as a general rule for susceptibility to GC. Likewise, short-time Dex-treatment did not modify the total GR expression level in thymocytes and splenocytes (Fig. 1B, e and g), which are known to be highly sensitive and relatively resistant to GC-induced apoptosis, respectively. To gain further insight on this issue, we exposed various leukemic cells to Dex and analysed its consequences on GR expression. Short-term incubations with Dex increased the GR level in the low responding CCRF-CEM ALL cells, and reduced the GR level in the GC-resistant NB4 PML and K562 CML cells, whereas no alteration was observed in the low responding BV173 CML and S49 T lymphoma cells (Fig. 1C). No further significant changes in GR expression were observed after 20 hrs of incubation with Dex (data not shown), suggesting that GR up and downregulation are early events occurring immediately following GC exposure. In CCRF-CEM cells, GR upregulation seems to be essential for reaching the threshold level required to turn on an apoptotic response. The latter may explain why the response to GC in these cells is delayed. This is in contrast to the highly GC-sensitive cells, such as PD1.6 and thymocytes, where the basal GR level is sufficiently high to allow commencement of the apoptotic process at an early time-point. The different regulation of GR expression by GC observed in each cell line may be due to different utilization of the 1A transcript. It is likely that the 1A transcript is predominant only in cells that respond to GC with GR upregulation, whereas the GC-refractory GR transcripts are the major ones in the others. Alternatively, the 1A transcript is already maximally utilized in these cells. Altogether, the above data show that the basal GR expression level per se is not an www.landesbioscience.com

index for susceptibility to GC-induced apoptosis, although low GR expression that is not upregulated by GC, acute downregulation of GR by GC (e.g., NB4, K562 and IM-9) or mutant GR (e.g., EL-4, Jurkat) are factors contributing to GC-resistance. These observations suggest that a better means to predict a positive GC response in newly diagnosed leukemia patients would be to measure the GR expression level after GC treatment rather than at the basal state. A better response is expected in cells where high GR level is maintained or where GR is upregulated by GC. Acute GR downregulation in response to GC has been observed in many non-hematopoietic cells (reviewed in ref. 28), in addition to the above-mentioned leukemia cases. The reasons for GR downregulation are poorly understood. Some data suggest that it is caused by degradation via the ubiquitin-proteasome system.29-31 GR downregulation is also a common obstacle of GC therapy as originally GC-sensitive tumors turn resistant upon repeated GC treatments.18,28 Thus, one approach to circumvent this predicament would be to combine GC therapy with a proteasome inhibitor such as bortezomib (Velcade), which is effective in the treatment of MM.32 On the other hand, some reports show that proteasomal inhibition may actually prevent GC-induced apoptosis.33,34 Hence, the effectiveness of proteasome inhibition in combination with GC therapy ought to be studied in detail. The antagonistic effects of anti-apoptotic proteins. Another important factor that prevents GC-induced apoptosis is the expression of anti-apoptotic proteins such as Bcl-2, Bcl-XL and Mcl-1. All of the GC-resistant cells analyzed by us express one or more of these proteins (Fig. 1A, b-d). The antagonistic effects of Bcl-2 and Bcl-XL on GC-induced apoptosis have been well described (reviewed in Almawi et al., ref. 35). The classical role of Bcl-2 is illustrated in Bcl-2-/- knockout (KO) mice, which show fulminant lymphoid apoptosis.36 Also, thymocytes deficient in Bcl-XL undergo premature apoptosis.37,38 These anti-apoptotic proteins counteract the proapoptotic Bax, Bak, Bim, Puma, Bad and tBid proteins that are downstream mediators of GC-induced apoptosis.35,39-42 The BH3-only proteins Bim, Puma, Bad and Bid act upstream to Bax and Bak.43,44 The role of Bax and Bak in this apoptotic process is demonstrated in thymocytes from double KO Bax-/-Bak-/- mice that are resistant to GC-induced apoptosis.40 Thymocytes from single KO Bax-/- or Bak-/- mice were still sensitive to GC,40 indicating that Bax and Bak may compensate for each other. Thymocytes from either single KO Bim-/- or Puma-/- mice show only partial impairment in GC-induced apoptosis,39,41,42 suggesting that these proteins partake in this death process, but act in an overlapping manner. Noxa42 and Bid45 deficiency had only marginal effect on thymocyte sensitivity to GC-induced apoptosis, indicating redundant functions with other pro-apoptotic proteins. In leukemic cells, the sensitivity to GC was directly correlated with Bim expression level, and elimination of Bim by siRNA partially abrogated the response to GC,46 pointing to a contributing, but not exclusive, role of Bim. We have analyzed the basal Bim expression level in various leukemia and lymphoma cells (Fig. 1A, h). Most of these cells express moderate to high Bim levels regardless of their sensitivity to GC-induced apoptosis. In some cell lines, such as BV173, CCRF-CEM and S49, Bim expression was upregulated after a 20-hrs incubation with 100 nM Dex, whereas in K562, NB4 and B10 cells no effect on Bim expression level was observed (unpublished data). Also, several other pro-apoptotic proteins of the Bcl-2 superfamily are expressed at various levels in both GC-sensitive and resistant cells (Fig. 1A, e–k). Thus, GC-resistance seems to be dominated by the expression of anti- apoptotic proteins, rather than by a lack of pro-apoptotic proteins. This suggests that it may be possible to sensitize GC-resistant lymphoid cells for

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GC-induced apoptosis by abrogating the antagonistic effects of anti-apoptotic proteins. A recent trend in experimental cancer therapy is to develop Bcl-2 inhibitors,47,48 which could also be beneficial in a combinatorial treatment with GC. Since the Bcl-2 activity is affected by phosphorylation,49 it would be reasonable to apply various kinase activators or inhibitors that may ultimately reduce the activity of the anti-apoptotic proteins. Antagonistic effects of certain protein kinases. Another obstacle in leukemia therapy is the activation of cell survival transduction pathways that antagonize GC-induced apoptosis. Signaling via MAPK kinase (MEK) and extracellular signal-regulated kinase (ERK) antagonizes Dex-induced apoptosis.50 The MAP/MAPK and phosphatidylinositol 3-kinase (PI3K) signaling pathways are constitutively activated in the majority of AML cases.51,52 Also, other protein tyrosine kinases are deregulated in hematological malignancies,53 which in turn activate the Ras/Raf/MEK, NFκB, PI3K/AKT and β-catenin survival pathways. Thus, a favorable approach for GC therapy of GC-resistant leukemias and lymphomas would be to inhibit the relevant activated kinases that antagonize GC-induced apoptosis. For instance, GCresistance could be overcome in a MM cell line by cotreatment with the multi-targeted tyrosine kinase inhibitor CHIR-258.54 Akt inhibitors increased the sensitivity of a follicular lymphoma cell line to Dex-induced apoptosis by inducing Bad translocation to the mitochondria.55 However, one should keep in mind that excessive activation of a pro-survival kinase may eventually lead to cell death. This was demonstrated by Ft1-mediated activation of AKT, which dramatically increased Dex-induced apoptosis in T lymphocytes through increased production of the pro-apoptotic Fas ligand.56 GC-induced apoptosis could also be augmented by Rapamycin through inhibition of c-Jun N-terminal kinase (JNK).57

GENOMIC EFFECTS OF GC

We emphasized above the importance of sufficient GR expression for bestowing susceptibility to GC-induced apoptosis. However, it is still unclear how activation of GR triggers the apoptotic process. Structurally, the full-length human GRα consists of 777 amino acids (aa), which may be divided into subdomains according to their functions (Fig. 2A). GRα contains two transactivation domains, τ1 at the N-terminus (aa 77-262) and τ2 at the C-terminus (aa 527-556 and aa 753-768), a zinc-finger DNA-binding domain (DBD, aa 420-488) and a C-terminal ligand-binding domain (LBD, aa 556-777).28,58-60 The DBD consists of two highly conserved zinc fingers (Fig. 2B), which are crucial for the binding to GRE sequences. In addition, the DBD encodes domains necessary for binding of AP-1 and NFκB,61-63 a nuclear export signal (NES, aa 442-456),64 and a region mediating receptor dimerization (aa 458-462).65,66 The LBD binds GC as well as heat-shock proteins (HSPs)67 and is also involved in receptor dimerization.58 A lysine-rich nuclear localization signal (NLS1) is located adjacent to DBD that corresponds to aa 491-498 of human GR, which resembles the NLS of SV40 T antigen and interacts with importins α/β and 768 (Fig. 2A). Also, within the DBD there are two additional lysine- and arginine-rich regions (aa 467-471/aa 477-480) that contribute to the nuclear localization of GR19 (Fig. 2B). There is evidence for another NLS at the C-terminus, which is poorly defined.68,69 We have recently identified a mitochondrial localization signal (MLS) within aa 558-580 of human GR, which is the amphipathic α-helix 3 of LBD.19 In the absence of a ligand, GR is retained inactive in the cytosol, sequestered to a large heteromeric complex of HSPs and immunophilins,70 or to 14-3-3σ.71 Upon ligand-binding, GR dissociates 1020

from these complexes, thereby exposing its nuclear localization signals (NLSs) with subsequent translocation to the nucleus where it transactivates and transrepresses multiple genes.9,59,72-74 Transactivation occurs through interaction with GREs, whereas transrepression is attained through tethering transcription factors such as AP-1 and NFκB, or binding to negative GREs.74-77 It is still obscure which of these alterations in gene expression are crucial for the apoptotic effect of GC. Altogether, it seems that GC shifts the balance between pro-survival and pro-apoptotic gene expression.6 Most of these changes, however, occur both in GC-sensitive and GC-resistant cells. Much effort has been devoted to reveal genes affected by GC that may be responsible for the induction of the apoptotic response.9,78-84 However, it has been difficult to pinpoint which of these genes are indeed important in this regard. Some differences have been observed in the spectrum of genes affected by GC in GC-sensitive versus GC-resistant cells.85 Noteworthy are the upregulation of various isoforms of the pro-apoptotic proteins Bim46,84,86 and Puma87 and the downregulation of the Cdk2 inhibitor p27kip1 88 and the pro-survival gene c-Myc.89,90 Other relevant genes upregulated by GC are the glucocorticoid-induced leucine zipper (GILZ),91 the NFκB inhibitor IκB,92,93 T-cell death-associated gene 8 (tdag8),94 thioredoxininteracting protein (txnip)95 and granzyme A.96 The increase in IκB expression further depresses the NFκB pathway, which is also antagonized by direct interaction between NFκB and GR.62,63 Conflicting data have been published concerning GILZ. On the one hand, the number of CD4+8+ cells in the thymus was decreased in GILZ transgenic mice with a concomitant decrease in Bcl-XL level, suggesting a pro-apoptotic function of GILZ.91 On the other hand, blocking GILZ expression in CTLL-2 T cells by siRNA, increased the cell’s sensitivity to GC-induced apoptosis, suggesting that GILZ rather offsets Dex-induced apoptosis.97 Also, the role of tdag8 is elusive as thymocytes from tdag8 KO mice show normal sensitivity to GC-induced apoptosis.98 At the bottom line, GC modulates the expression of several genes that may act in concert to affect the apoptotic process. However, the mere expression of the pro-apoptotic genes is insufficient for eliciting an apoptotic response as these are usually kept in an inactive state.99 Further post-transcriptional modifications and protein-protein interactions are required for triggering the proapoptotic function of these proteins.43,99-101 Thus, transactivation, although necessary, is insufficient to elicit the apoptotic process. A current prevailing hypothesis is the possible role of GC-mediated transrepression in the induction of apoptosis. This was suggested in light of the central role of AP-1 and NFκB in controlling cell survival.7,74,77 Support for such a mechanism comes from the observation that a GR mutant (GR-LS7) compromised in transactivation, but normal in transrepression, is as effective as the wild-type receptor in inducing apoptosis.102 However, a dimerization-defective GR (GRdim) is unable to mediate GC-induced apoptosis although it retains competence for transrepression through interaction with AP-1 and NFκB.66 Thus, interference with AP-1 and NFκB per se is insufficient for inducing apoptosis. The role of transrepression is further questioned as a GR mutant deficient in transrepression is still able to induce apoptosis.62 Because dimerization is required for DNA-binding,66 it was suggested that DNA-binding is a prerequisite for eliciting the apoptotic response. However, a GR variant defective in DNA binding, which is mainly located in the cytoplasm, also induces apoptosis.103 Collectively, these data suggest that GC may induce apoptosis by a non-genomic pathway, which is likely to act in concert with the nuclear GR effects.

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GC-sensitive ones. Src is released from Hsp-complexes simultaneously with the release of GC-bound GR.111 However, the mechanism by which Src promotes apoptosis, is unknown. Cdk2 activation in thymocytes occurs at a later phase after Dex treatment and correlates with the onset of apoptosis.88 Caspase-8 activation and Bid cleavage to tBid occur downstream to Cdk2 activation, but upstream to mitochondrial dysfunction, suggesting a role of Cdk2 in regulating these processes.121 Cdk2 is known as a central regulator of the cell cycle. However, recent data shows that it also plays a role in distinct pathways of apoptosis.122 It has further been shown that overexpression of Bax accelerates Cdk2 activation following Dex treatment, whereas Bcl-2 overexpression has an opposite effect.88 This may be one of the mechanisms by which Bcl-2 prevents apoptosis. Excessive Cdk2 activation seems to be a turning point Figure 2. Schematic structures of human GRα (A) and DBD (B). τ1 and τ2 are transactivation domains. DBD leading the cells into the apoptotic is the DNA binding domain, and LBD the ligand binding domain. The arginine and lysine-rich nuclear localization signal (NLS; in dark green) is partially located within the DBD, and stretches to a C-terminally region pathway. The central question is which adjacent to DBD. A nuclear export signal (NES; in bright green) is defined to the region between the two signals delivered by GR activate Cdk2? zinc-fingers of the DBD. An amphipathic mitochondrial localization signal (MLS; in pink) comprises the A decrease in p27kip1 expression level is α-helix 3 of LBD. The MLS contains positively charged (in red), hydrophilic (in orange) and hydrophobic (in observed in thymocytes during the first blue) amino acids, but no negatively charged residues. An amino-acid stretch important for dimerization is hours of exposure to Dex.88 But the found within the second zinc-finger, also termed the D-loop. (C) The mitochondrial GR is localized to the mere reduction in p27kip1 expression is inner part of the mitochondria. Isolated mitochondria from Dex-treated PD1.6 cells were either washed in 148 insufficient for activating Cdk2,88 indiprior mitochondria buffer without detergent, or with 0.1% NP-40, 0.25% or 0.5% digitonin as described cating that additional signals should be to lysis in protein sample buffer and Western blotting using PA1-511A antibodies to GR (a), PC548 antibodies to the voltage-dependent anion channel (VDAC; Oncogene Research Products), which is located delivered by GR. within the outer mitochondrial membrane (b); and FL-298 antibodies to adenine nucleotide translocator Some other non-genomic effects of (ANT; Santa Cruz Biotech.), which is located within the inner mitochondrial membrane (c). The outer mitoGC are mediated by a membrane chondrial membrane was disrupted with 0.5% digitonin verified by the disappearance of VDAC (lane 4). form of GR, which is expressed on a GR was still found in the remaining mitoplast after this treatment, indicating that it is mainly localized to the limited number of hematopoietic cells inner part of the mitochondria. such as monocytes, the macrophage cell line RAW264.7 and S49 T lymphoma cell variants,19,20,123 as well as on certain non-hematopoietic NON-GENOMIC EFFECTS OF GC cells.124,125 Since the membrane GR has been associated with the 1A Several rapid effects have been described to occur within a short transcript and a cDNA derived from the 1A transcript induced time frame after GC treatment, which cannot be related to the apoptosis, it was anticipated that membrane GR is involved in apop104-107 genomic effects of GC. These include generation of reactive 20 oxygen species (ROS); activation of phosphatidylinositol-specific tosis. However, this cDNA gave also raise to high GR level in the phospholipase C (PI-PLC) with subsequent transient calcium mobi- cytoplasm, which could be the trigger of apoptosis. No comparison lization; activation of acidic sphingomyelinase leading to increased was made with a GR cDNA that was unable to produce membrane hematopoietic cells, e.g., T cells, do not display memceramide generation; and lysosomal release of cathepsin B.1,34,107-111 GR. Some 123 brane GR, despite transcribing the 1A transcript. Thus, the Moreover, GR becomes hyperphosphorylated immediately after expression of membrane GR seems to be dictated by another factor. 112,113 binding of its ligand, indicating that one or more kinases are is a correlation between membrane GR find out whether there To activated at this stage. Although several kinases such as cyclin-depenand apoptotic susceptibility to GC, we studied a series of lymphoid dent kinase (CDK), JNK, ERK and glycogen synthase kinase-3 (GSK3) 19 have been shown to phosphorylate GR,113-115 it is unknown which cells showing differential response to GC. We observed membrane kinases activated by Dex phosphorylate GR. GR phosphorylation GR expression on some GC-resistant cells, but not on the highly 19 modulates GR transactivation activity,113-115 affects the protein GC-sensitive PD1.6 and 2B4 cells. Its expression was not upregu116 117-120 lated by Dex treatment in the latter cells. Thus, membrane GR is not and nucleocytoplasmic shuttling. However, its stability apoptosis, and its mere presence does not for GC-induced required influence on the apoptotic ability of GR is unclear. By using inhibitors to Src and Cdk2, it has been suggested that impose susceptibility to GC. Other studies also did not find any coractivation of these kinases is essential for GC-induced apoptosis of relation between membrane GR expression on ALL cells and in vitro thymocytes.88,111,121 A central question is whether GC activates these sensitivity to GC.5 So the question remains how GR mediates the kinases in both GC-sensitive and GC-resistant cells, or only in non-genomic effects required for eliciting apoptosis. www.landesbioscience.com

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ROLE OF MITOCHONDRIAL GR IN ELICITING APOPTOSIS In light of the central role of mitochondria in both integrating pro- and anti-apoptotic signals and in GC-induced apoptosis, it was plausible to investigate whether GR may exert some of its nongenomic effects in this intracellular organelle. GR has been detected within the mitochondria in several cell types.126-130 In order to study the role of mitochondrial GR in GC-induced apoptosis, we compared the ability of GR to translocate to the mitochondria in various T lymphoid cell lines, which varied in their sensitivity to GC.19 Interestingly, mitochondrial translocation of GR was observed only in cells sensitive to GC-induced apoptosis, but not in GC-resistant ones. In contrast, nuclear translocation occurs in both cell types. This finding is the first qualitative difference in GR behavior described that may explain the differential response of lymphoma cells to GC therapy. Further support for a role of mitochondrial GR in mediating apoptosis comes from the observation that thymic epithelial cells, which trigger apoptosis of PD1.6 cells in a GRdependent manner,18 induce GR translocation to the mitochondria, but not to the nucleus.19 Thus, GR translocation to the mitochondria may act independently of the nuclear GR effects. To further demonstrate that GR may induce apoptosis when localized in the mitochondria, we constructed a GR directed to the mitochondria by adding the strong mitochondrial localization signal (MLS) of cytochrome C oxidase N-terminally to the GFP-GR fusion protein. This GR was able to induce apoptosis in a GC-independent manner,19 indicating that exclusive expression of GR in the mitochondria is sufficient for inducing apoptosis. Further studies are required to elucidate the mechanisms by which mitochondrial GR elicits apoptosis. Scheller et al.127,128 demonstrated by immunoelectron microscopy that GR is located to the inner mitochondrial membranes and in the inner matrix space. We have also noticed by sub-mitochondrial fractionation analysis that the GR is found mainly in the inner part of the mitochondria (Fig. 2C). GRE elements have been detected in the mitochondrial genome,131 which may explain some of the GC effects on mitochondrial transcription and energy metabolism.128,132,133 Dex-treatment did not reduce the ATP level in a GC-sensitive thymic lymphoma prior to the onset of apoptosis,133 thereby excluding ATP deprivation as an underlying cause. Some mitochondrial metabolic differences have been observed in GC-sensitive vs. resistant cells.133 Most notable is the reduction in hexokinase activity within the mitochondria of Dex-treated thymic lymphoma cells.133 The GC-sensitive cells showed lower activity of this enzyme than the resistant ones,133 and overexpression of hexokinase II blocked Dex-induced apoptosis.134 GC may have a direct effect on the mitochondrial hexokinase activity, or it may act indirectly via the GC-induced transrepression of the hexokinase gene.82 Interestingly, hexokinase was shown to bind to voltage-dependent anion channel (VDAC) and interfere with the ability of Bax to associate with the mitochondria.135 These data suggest that a reduction in hexokinase level, such as that caused by GC, may facilitate Bax binding to VDAC. Thus, some of the mitochondrial metabolic effects of GC may modulate GC-induced apoptosis. However, it is likely that GR has additional effects in the mitochondria. The translocation of GR to the mitochondria in the GC-sensitive cells occurs within minutes after exposure to Dex and parallels nuclear GR translocation.19 It has been postulated that GR binding to mitochondrial membranes may directly regulate its membrane potential (∆Ψm ).127,136 We observed a lower intensity of mitotracker staining in transfected cells expressing mitochondrial GR than in the 1022

non-transfected cells of the same sample.19 However, a longitudinal follow up of the changes in membrane potential using mitotracker staining of Dex-treated thymic lymphoma cells, indicated that a fall in ∆Ψm is a relative late event18 and can thus not be explained by the mere binding of GR to mitochondrial membranes which occurs immediately after Dex exposure. It could rather be that the rapid translocation of GR to the mitochondria accounts for some of the early non-genomic effects of GC, such as ceramide and ROS generation as well as the transient Ca2+ mobilization, which are crucial for initiating the apoptotic response. Several lines of evidence support the concept that mitochondria play a central role in regulating these non-genomic effects. Firstly, a mitochondrial sphingomyelin pool has been shown to produce ceramide within the mitochondria,137 and ceramide activates mitochondrial PP2A, which in turn dephosphorylates Bcl-2 with subsequent cell death.138 Secondly, the mitochondria are a major source of ROS production,139 and in Dex-treated thymocytes, an increase in hydrogen peroxide (H2O2) production occurs at the ubiquinone cycle at complex III of the mitochondria.33 Thirdly, the mitochondria regulate calcium homeostasis,140 and a cross-talk between the mitochondria and the endoplasmic reticulum, another intracellular Ca2+ store, has been proposed.141 Altogether, the mitochondria are involved in both initiating and propagating death signals.141 It is therefore likely to assume that the rapid and sustained increase in the mitochondrial GR expression level occurring in GC-sensitive cells after Dex treatment may influence some of these processes. GR is not the first protein to be detected that exerts its apoptotic function in the mitochondria. Both the tumor-suppressor p53 and the orphan receptor Nur77, act directly on the mitochondria besides regulating gene transcription.142,143 p53 has been shown to interact with both Bcl-2 and Bcl-XL in the mitochondria,142 and Nur77 with Bcl-2.143 Further, it has been shown that Bcl-XL sequesters cytoplasmic p53 after genotoxic stress, and only after p53-induced Puma expression, p53 dissociates from Bcl-XL to directly activate Bak and Bax.144 Thus, the non-genomic pro-apoptotic effects of p53 are tightly coupled with its genomic effects. Nur77 has even been shown to convert Bcl-2 from an anti-apoptotic to a pro-apoptotic protein.143 The latter effect explains why Bcl-2 is unable to counteract Nur77mediated apoptosis. An implication of this finding is that a favorable approach to overcome GC-resistance in Bcl-2 overexpressing cells would be to combine GC with a Nur77-inducing agent. So far it seems unlikely that GC-induced apoptosis is mediated through an interaction between GR with Bcl-2 or Bcl-XL, as highly GC-sensitive cells barely express these proteins. Moreover, mitochondrial GR translocation is prevented in Bcl-2 and Bcl-XL overexpressing cells, which may actually be one of the mechanisms by which these proteins antagonize GC-induced apoptosis. Whether GR has any direct effect on Bax or Bak activation as seen with p53,145,146 is a matter for further research.

CONCLUDING REMARKS

GC-induced apoptosis is a complex process tightly regulated by multiple genes, proteins and signaling pathways (Fig. 3). The fact that cycloheximide and actinomycin D abrogate this process, suggests a requirement for de novo protein synthesis.88,147 Although the genomic effects of GC are important for tipping the balance between pro-survival and pro-apoptotic proteins, non-genomic effects are crucial for triggering the apoptotic signals. Some of these are mediated by protein kinases dissociated from the HSP-complexes together with

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Figure 3. A model of GC-induced apoptosis. In the absence of ligand (GC), GR is sequestered in the cytoplasm to a large heteromeric Hsp complex, which also binds various kinases including Src. Upon GC binding (1), GR and Src dissociate from the Hsp complex (2), and GR dimerizes and translocates to the nucleus or the mitochondria (3). GR translocation to the mitochondria occurs in GC-sensitive, but not in GC-resistant lymphoid cells, whereas nuclear GR translocation occurs in both cell types. The mitochondrial GR may be the mediator of some of the early nongenomic effects (4), which are essential for initiating the apoptotic process. It may also directly affect mitochondrial functions. The nuclear GR causes both transactivation (5) and transrepression (6). Important upregulated genes are IκB, which inactivates NF-κB in the cytoplasm, and the pro-apoptotic Bim and Puma that act upstream to Bak and Bax. The mere expression of Bim and Puma is insufficient to elicit apoptosis. These proteins need to be activated post-transcriptionally. It is still uncertain which factors induced by GR activates these proteins. It is likely that the early production of ROS and ceramide as well as calcium may activate these proteins in combination with a kinase (7). As inhibition of Src prevents the apoptotic process, it could be that this kinase or a kinase affected by Src modulates the activity of the pro-apoptotic proteins (7). Activation of Bim and Puma leads to the activation of Bax and Bak, which undergo conformational changes and translocate to the mitochondria where they affect the mitochondrial membrane potential ∆Ψm (8). Important genes transrepressed by GR include hexokinase and c-Myc. Hexokinase prevents Bax binding to VDAC and c-Myc is a pro-survival protein. Thus, reduction in their expression levels makes the cells more prone to apoptosis. The expression level of the Cdk2 inhibitor p27kip is dramatically reduced by Dex, which could be due to transrepression and/or accelerated degradation. Together with an additional signal, Cdk2 becomes activated (9), leading to the activation of caspase 8 and cleavage of Bid into tBid (9). tBid translocates to the mitochondria (9), where it cooperates with Bax and Bak to cause the release of cytochrome C (CytoC) and SMAC/DIABLO (10). CytoC forms the apoptosome with Apaf-1 and procaspase-9 and turns on the executive caspase cascade (11). SMAC neutralizes the inhibitor of apoptosis proteins (IAPs), thus having a potentiating effect on the caspase cascade, eventually leading to apoptosis.

GR upon GC binding. But more important is the mitochondrial translocation of GR observed in GC-sensitive cells. GR in the mitochondria is both essential and sufficient for inducing apoptosis.19 It is likely that the mitochondrial GR cooperates with the nuclear GR in GC-induced apoptosis, thereby reminiscing p53 and Nur77mediated apoptosis, which both are propagated by coordinated effects on nucleus and mitochondria. The observations that mitochondrial GR may induce apoptosis independently of the nuclear GR effects, and that mitochondrial and nuclear translocations are differentially regulated,19 indicate that it should be possible to induce apoptosis of leukemia cells by redirecting the GR to the www.landesbioscience.com

mitochondria. Such an approach is especially attractive given the multiple adverse effects caused by the nuclear GR. Acknowledgements

This work was supported by The Concern Foundation, CA and The Israel Cancer Association. References 1. Greenstein S, Ghias K, Krett NL, Rosen ST. Mechanisms of glucocorticoid-mediated apoptosis in hematological malignancies. Clin Cancer Res 2002; 8:1681-94. 2. Haarman EG, Kaspers GJ, Veerman AJ. Glucocorticoid resistance in childhood leukaemia: Mechanisms and modulation. Br J Haematol 2003; 120:919-29. 3. Rajkumar SV, Gertz MA, Kyle RA, Greipp PR. Current therapy for multiple myeloma. Mayo Clin Proc 2002; 77:813-22.

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