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RCAN1-4 knockdown attenuates cell growth through the inhibition of Ras signaling
FEBS Letters 583 (2009) 2557–2564
journal homepage: www.FEBSLetters.org
RCAN1-4 knockdown attenuates cell growth through the inhibition of Ras signaling Hong Joon Lee a,b, Young Sun Kim a,b, Yasufumi Sato c, Young-Jin Cho a,b,* a b c
Department of Pharmacology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea Cell Death Disease Center of MRC, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea Department of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan
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Article history: Received 25 May 2009 Revised 12 June 2009 Accepted 11 July 2009 Available online 18 July 2009 Edited by Lukas Huber Keywords: RCAN1/DSCR1 Cell growth Ras ERK/MAPK Translation eIF4E
a b s t r a c t Forced changes in the expression of regulator of calcineurin 1 (RCAN1) affects cell growth. This has been linked to the suppression of calcineurin-nuclear factor of activated T cells signaling by RCAN1. Here, we describe a novel role of RCAN1 isoform 4 in proper expression of Ras protein and its signaling. RCAN1 isoform 4 knockdown attenuated growth factor-induced extracellular signal-regulated kinase activation and cell growth; reduced Ras levels and its translation rate; and led to a reduction of eukaryotic initiation factor 4E in the initiation complex and a slight repression of global protein synthesis. Experiments utilizing activity-modified mutants of calcineurin A demonstrated that these effects were calcineurin-independent. Our findings reveal a previously unknown role of RCAN1-4 in protein synthesis, which may be relevant to cell growth. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The human regulator of calcineurin 1 (RCAN1) gene, also termed Down syndrome candidate region 1, was first isolated near the portion of the Down Syndrome Critical Region on chromosome 21 [1]. The gene consists of seven exons; exons 1–4 can be alternatively spliced, resulting in four different transcripts denoted as RCAN1-1 through RCAN1-4. RCAN1 protein binds to the catalytic subunit of a Ca2+/calmodulin-dependent phosphatase calcineurin and inhibits its function [2]. Elevated RCAN1 expression has been observed in neurodegenerative diseases, including Alzheimer’s disease and Down syndrome [3], in the heart after pressure-overload [4], and in the peri-infarct cortex following experimental stroke [5]. Altered RCAN1 expression exerts diverse influences on cell proliferation. Over-expression of hamster homologue adapt78 induces G1-phase growth arrest in hemagglutinin (HA)-1 cells [6]. Consistently, adenovirus-mediated introduction of RCAN1-4 into endoAbbreviations: RCAN1, regulator of calcineurin 1; NFAT, nuclear factor of activated T cells; ERK, extracellular signal-regulated kinase; eIF4E, eukaryotic initiation factor 4E; GFP, green fluorescent protein; HA, hemagglutinin; GST, glutathione S-transferase * Corresponding authors. Address: Department of Pharmacology, College of Medicine, The Catholic University of Korea, Banpo-dong 505, Seocho-gu, Seoul 137701, Republic of Korea. Fax: +82 2 536 2485. E-mail address: [email protected] (Y.-J. Cho).
thelial cells induces G0/G1 arrest and inhibits angiogenesis [7]. On the other hand, the over-expression of RCAN1-1 promotes the proliferation of PC-12 cells [8]. In a study addressing isoform-specific functions, over-expression of RCAN1-1 induced endothelial cell proliferation, but RCAN1-1-specific knockdown did not affect the baseline cell growth. On the other hand, over-expression of RCAN1-4 did not affect baseline endothelial cell growth, and RCAN1-4-specific knockdown enhanced cell proliferation [9]. These observations suggest that RCAN1 may play differential roles in cell growth, dependent on cell types and isoforms of RCAN1. Until now, the speculated role of RCAN1 in cell growth has been mainly based on its function as a negative regulator of calcineurin signaling. Hence, the effects on growth-related signal transduction molecules have not been elucidated in detail. Here, we investigated the effects of altered RCAN1-4 expression on cell proliferation and growth factor-induced activation of extracellular signal-regulated kinase (ERK) pathway.
2. Materials and methods 2.1. Cell culture Human U87MG glioblastoma cells and human 293T embryonic kidney cancer cells were obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in minimal
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.07.023
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essential medium (U87) and Dulbecco’s modified Eagle’s medium (293T) supplemented with 1.0 mM sodium pyruvate and 10% fetal bovine serum under an atmosphere of 5% CO2 at 37 °C. If needed, the cell number was counted using a Z2 Coulter Counter (Multisizer II, Beckman–Coulter). 2.2. Biochemical procedures Chemicals and antibodies, plasmid construction, short interfering RNA (siRNA)-mediated RNA interference, metabolic cell labeling and immunoprecipitation, pulse-chase analysis, pull-down assays, luciferase assay, Triton X- 114 separation, real-time quantitative RT-PCR analysis, and immunoblot analysis were performed according to standard protocols. Details can be found in the Supplementary data. 3. Results 3.1. RCAN1-4 knockdown suppresses U87MG cell growth We compared the growth rate of U87MG cells with RCAN1-4 knocked down and control cells. To suppress the expression of endogenous RCAN1-4, we designed siRNA targeted against exon 4 (siRCAN1-4). Transfection with this siRNA resulted in an approximate 90% reduction in mRNA levels, which were quantified by real-time RT-PCR analysis (Fig. 1A). After transfection with siRCAN1-4 or control siRNA, the number of cells in culture was counted every 24 h until 96 h. The cell numbers for siRCAN1-4transfected and control cultures were not significantly different 24 and 48 h after the transfection. However, at 72 h and 96 h, the growth of siRCAN1-4-transfected cells was significantly retarded relative to control cells (29% and 35%, respectively; Fig. 1B). Earlier reports demonstrated the arrest of cell cycle progression in RCAN1modulated cells [6,7]. To investigate the involvement of cyclindependent kinase inhibitors in the retardation of cell growth, we examined the level of p21 and p27Kip1. As shown in Fig. 1C, siRCAN1-4 significantly increased p21 and p27 Kip1 expression in a time-dependent manner. 3.2. RCAN1-4 knockdown attenuates the growth factor-induced ERK activation Next, we examined ERK activation in response to growth factors. Compared with controls, siRCAN1-4-transfected cells exhibited an attenuated induction of ERK phosphorylation following bFGF treatment (Fig. 2A). Attenuated ERK phosphorylation was also observed in response to phorbol 12-myristate 13-acetate and calcium ionophore A23187 stimulation (data not shown). We examined the activation of the upstream ERK signal to identify a target molecule that is directly inhibited by RCAN1-4 knockdown. Transfection with siRCAN1-4 attenuated the EGF-induced phosphorylation of Raf-1 and MEK (Fig. 2B). Notably, the level of Ras protein was also reduced in siRCAN1-4-transfected cells relative to controls. Transcription of the reporter gene mediated by Elk-1, a downstream effector molecule of ERK, was also reduced in siRCAN1-4-transfected cells (Fig. 2C). Reciprocally, vector-mediated over-expression of green fluorescent protein (GFP)-RCAN1-4 partially rescued the attenuated phosphorylation of ERK in siRCAN14-transfected cells (Fig. 2D). 3.3. RCAN1-4 knockdown reduces steady-state levels of Ras protein Based on the reduction of Ras expression, we hypothesized that the attenuated response of the ERK pathway to growth fac-
Fig. 1. RCAN1 knockdown suppresses U87MG cell growth. (A) Real-time PCR for RCAN1-4 (left) and detection of RCAN1-4 expression (right) in U87MG cells transfected with either siRCAN1-4 or siCon. (B) U87MG cells were transfected with either a specific siRNA for RCAN1 isoform 4 (siRCAN1-4) or control siRNA (siCon), and cell growth was determined by a Coulter counter. Results are the means and S.D. of three independent experiments (*P < 0.05, **P < 0.001 versus time-matched control). (C) Representative immunoblot showing the time-dependent change of p21 and p27Kip1 expressions following transfection with either siRCAN1-4 or siCon.
tors resulted from a deficit of Ras-mediated signal transduction. We examined Ras activation following the stimulation of EGF. As expected, the amount of GTP-bound Ras in siRCAN1-4-transfected cells was significantly low compared to control (by 42% and 31% at 10 min and 30 min after stimulation, respectively; Fig. 3A). We then investigated whether there is a change in the synthesis of Ras and its post-translational prenylation, which is important for Ras protein localization to the plasma membrane and receiving signals from receptors [10]. The cytosolic and membrane-bound forms of Ras were separated by Triton X-114 and quantified by Western blot analysis [11]. Prenylated Ras protein in detergent phase was reduced by 47% in siRCAN1-4-transfected cells compared to control (Fig. 3B). A longer exposure of
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Fig. 2. RCAN1-4 knockdown attenuates growth factor-induced ERK activation. (A) Time course study showing bFGF (10 ng/ml)-induced ERK phosphorylation (top) in U87MG cells transfected with either siRCAN1-4 or siCon. Densitometric ratios of the p-ERK/ERK signal (bottom) are represented as mean and S.D. from three independent immunoblots (*P < 0.05 versus time-matched control). (B) Immunoblot showing EGF (20 ng/ml for 10 min) induced phosphorylation of signaling molecules in an upstream ERK pathway of 293T cells. (C) Attenuation of Elk-1 promoter-driven luciferase expression following EGF treatment (20 ng/ml for 4 h) in siRCAN1-4-transfected 293T cells. Results are the means and S.D. of four independent experiments. *P < 0.05 compared with siCon. (D) Immunoblot of EGF (20 ng/ml for 10 min) induced phosphorylation of ERK in 293T cells co-transfected with either a vector encoding RCAN1-4 (GFP-RCAN1) or GFP, and either siRCAN1-4 or siCon in the combinations indicated.
the same panel showed that non-prenylated Ras in aqueous phase was also reduced by 45%. These observations suggest that siRCAN1-4 down-regulates the overall level of Ras protein expression rather than a post-translational maturation process. Consistent with the ERK response to EGF (Fig. 2C), over-expression of GFP-RCAN1-4 partially rescued Ras expression (Fig. 3C). We next examined an isoform-specific effect of RCAN1. Cells were transfected with siRCAN1-4 and siRCAN1-1 specifically targeted to exon1. Transfection of siRCAN1-1 inhibited Ras expression by 17% (Fig. 3D). Interestingly, transfection of siRCAN1-1 increased the transcription of endogenous RCAN1-4 by twofold (data not shown). The transcription of RCAN1-4 gene is activated potently by calcineurin due to the presence of multiple consensus nuclear factor of activated T cells (NFAT) binding sites within an internal promoter region [12]. Thus this increase of RCAN1-4 transcript is probably due to the release of calcineurin from an inhibitory effect of RCAN1-1. In the next experiments, we used only RCAN1-4.
3.4. RCAN1-4 knockdown does not affect Ras transcription and protein stability The cellular protein level is the net result of protein synthesis and degradation. To explore the molecular basis of the reduction in Ras expression, we first examined transcriptional level of Ras isoforms. Real-time PCR analysis revealed that H- and K-Ras mRNA levels were not significantly decreased, and N-Ras transcription was rather increased in siRCAN1-4-transfected cells (Fig. 4A). These results suggest that reduced expression of Ras protein was not due to suppression in transcriptional stage. To investigate the stability of Ras protein, we next examined whether it is actively degraded by various cellular proteases. siRCAN1-4-transfected or control cells were treated for 8 h with the proteasomal inhibitor MG132 and the lysosomotrophic agent chloroquine. The cells were then subjected to immunoblot analysis for Ras. Inhibition of these proteases did not block the reduction of Ras expression in siRCAN1-4-transfected cells (Fig. 4B). To confirm that siRCAN1-4 does
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Fig. 3. RCAN1-4 knockdown reduces the steady-state level of Ras protein. (A) Time course study showing the formation of GTP-bound Ras (GTP-Ras) after EGF treatment (20 ng/ml) to 293T cells transfected with indicated siRNA, which was assayed by pull-down (PD) with glutathione S-transferase-RBD. (B) Immunoblot showing the attenuated expression of prenylated (upper bands) and non-prenylated Ras (lower bands) in siRCAN1-4-transfected 293T cells. a, aqueous phase; d, Triton X-114 detergent phase containing lipid-associated proteins. The relative density of bands was given below. The value obtained in siCon-transfected cells were set as 100% (C) Immunoblot showing that co-transfection of GFP-RCAN1-4 increases Ras expression in 293T cells transfected with siRCAN1-4. (D) Immunoblot showing effects of isoform-specific siRCAN1s on Ras expression in U87MG cells.
not affect Ras protein stability, we performed a pulse-chase analysis for Ras degradation. The cells were pulsed with [35S]-methionine/cysteine for 4 h and then chased for 20 h. Quantitative analysis of the remaining radiolabeled Ras revealed that the degradation rates of Ras protein were not significantly different between the siRCAN1-4-transfected cells and controls (Fig. 4C). Collectively, these results indicate that the reduction in Ras expression is not due to enhanced protein degradation or attenuated transcription.
(eIF4E-BP1) levels. To determine whether these alterations could disrupt the eIF4E complex, we examined the association of eIFs by pulling down with m7GTP-sepharose resin. The precipitation assay showed a reduced association of eIF4E and eIF4E-BP1, whereas the amounts of eIF4A and eIF4G were not significantly altered (Fig. 5C). These findings suggest an inhibitory role for RCAN1-4 in the translation initiation processes, probably by down-regulating eIF4E.
3.5. RCAN1-4 knockdown decreases Ras protein translation
3.6. siRCAN1-4 inhibits translation by a calcineurin-independent mechanism
We next examined the translational efficiency of Ras protein. The cells were cultured in a medium containing [35S]-methionine/cysteine to radiolabel the newly synthesized proteins for two time periods: 30 and 60 min. After the indicated incubation, cell lysates were obtained and analyzed by SDS–PAGE. Quantitative imaging with a phosphorimager demonstrated that newly synthesized Ras in siRCAN1-4-transfected cells was 64.6% and 74% less than that of time-matched controls at 30 and 60 min, respectively (Fig. 5A). Overall, 35S-labeled cellular protein was also reduced in RCAN1-4-transfected cells by about 20% relative to control cells (Fig. 5B). It is notable that Ras synthesis is more intensively inhibited than global translation. These observations suggest a role for RCAN1-4 in translational regulation. The eukaryotic initiation factors eIF4E, eIF4G, and eIF4A comprise the trimeric eIF4F complex, which promotes cap-dependent mRNA translation [13]. Therefore, we tested whether RCAN1-4 knockdown alters the expression of eIFs or assembly of the eIF4F complex. Transfection with siRCAN1-4 had no measurable effect on the levels of eIF4A and eIF4G proteins but resulted in reduced eIF4E and eIF4E-binding protein
RCAN1 is an endogenous inhibitor of calcineurin, and targeted deletion or knockdown of this gene results in the activation of calcineurin [9,14]. We next examined whether the elevated calcineurin activity in siRCAN1-4-transfected cells contributes to the attenuated translation of Ras protein. We knocked down either calcineurin Aa (siCnAa) or calcineurin Ab (siCnAb) simultaneously with RCAN1-4. Reducing the abundance of calcineurin did not rescue Ras expression in siRCAN1-4-transfected cells (Fig. 6A). Similarly, pharmacological inhibitors of calcineurin, cyclosporine A and FK506, did not restore the attenuated ERK response to bFGF (Fig. 6B). We then investigated whether the translation deficit in siRCAN1-4-transfected cells was also independent of calcineurin. We examined the translational efficiency in siCnA-transfected cells and siRCAN1-4-transfected cells by monitoring vector-mediated expression of GFP as a reporter [15,16]. The GFP expression vector was transfected 24 h after siRNA transfection, and cells were incubated another 24 h before immunoblot analysis. Consistent with the results of a metabolic labeling experiment for the analysis of
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Fig. 4. RCAN1-4 knockdown does not affect Ras transcription and protein stability. (A) Effects of siRCAN1-4 on the Ras transcript levels of 293T cells measured by realtime RT-PCR. *P < 0.05 compared with siCon. (B) Immunoblot showing that a proteasome inhibitor (MG132; 4 lM for 8 h) or lysosome inhibitor (chloroquine; 200 lV for 8 h) does not block the siRCAN1-4-induced reduction of Ras in U87MG cells. (C) Degradation of Ras protein. U87MG cells were pulsed with [35S]methionine/cysteine for 4 h. Radiolabeled Ras was chased for 20 h.
protein synthesis, the transfection of siRCAN1-4 significantly inhibited the expression of GFP (Fig. 6C). Interestingly, the siCnAs also inhibited GFP expression instead of an expected opposite effect to that of siRCAN1-4 (Fig. 6D). Next, we assessed whether calcineurin activation enhanced Ras and GFP expression. Transfection of the constitutively active form of calcineurin increased Ras and GFP expression by 10% and 32%, respectively, indicating a positive role of calcineurin A in Ras and GFP translation (Fig. 6E). Taken together, these data suggest that siRCAN does not inhibit Ras translation by augmenting calcineurin signaling. 4. Discussion The evidence that proper RCAN1 expression plays an important role in proliferation has been observed in diverse cell type. RCAN1 is involved in proliferation as well as growth suppression [6– 9,14,17]. Furthermore, each RCAN1 isoform plays differential roles in cell proliferation in response to a growth factor [9]. Thus, the
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mechanism by which RCAN1 modulates cell proliferation is complicated and not completely understood. In contrast with the previous report utilizing vascular endothelial cells [9], we demonstrated that RCAN1-4 knockdown results in the retarded growth of U87MG cells and the attenuated activation of the ERK pathway in response to growth factors. In addition, our results showed for the first time that RCAN1-4 knockdown suppressed translation initiation complex formation and total protein synthesis in a calcineurin-independent manner. This may affect cell growth because many growth regulators are encoded by weak mRNAs, translation of which is more sensitive to small perturbations in translation initiation complex formation [13,18–20]. Among them, this study showed Ras protein as one of the signaling molecules relevant to cell growth and sensitive to the defect of translation induced by knockdown of RCAN1-4. RCAN1-4 reportedly works as a negative regulator of cell growth by inhibiting calcineurin/NFAT pathway-dependent mechanism [7,9]. Thus, RCAN1-4 knockdown can enhance cell growth via a calcineurin-dependent mechanism [9], and can also suppress cell growth via a calcineurin-independent mechanism, which is suggested by this study. The dominance between these opposite events may be affected by the temporal status and types of cells. In addition to this, isoform-specific differential effects of RCAN1 on calcineurin may explain the diversity of growth responses to RCAN1 modulation. In addition, the phosphorylation status of RCAN1 is reported to affect calcineurin signaling [21]. Thus, the overall effects of RCAN1 on growth would be extremely diverse depending on the cellular context. The independence of siRCAN1-4-induced down-regulation of Ras on the calcineurin pathway was shown in several experiments utilizing pharmacological inhibitors, siRNAs, and calcineurin A mutants. Although a mechanism was not addressed in this study, these experiments revealed that calcineurin is a positive regulator of Ras translation in U87MG cells. Similarly, a requirement for calcineurin in translational machinery activation has been reported in rat pancreatic acini [22]. Given that RCAN1-4 is a negative regulator of calcineurin, RCAN1-4 knockdown is expected to exert influence on Ras expression into two modes: facilitation of translation by activating a calcineurin-dependent mechanism and translation inhibition via a calcineurin-independent mechanism. The mechanism by which RCAN1-4 knockdown preferentially inhibits the translation of Ras protein is not completely solved in this study. In this respect, we showed that knocking down RCAN1-4 reduced eIF4E expression and its binding to the 7methyl-GTP cap structure. In eukaryotes, roughly 90% of protein is synthesized through cap-dependent translation. Indeed, knocking down RCAN1-4 reduced global translation in U87MG cells. This observation raised the question of why Ras expression was strongly inhibited compared to that of other proteins observed in this study. Cellular mRNAs differ hugely in the amount of eIF4F complex required for efficient translation [13]. The translation efficiency of mRNA with highly complex 50 -untranslated regions (50 UTR) is especially dependent on eIF4E levels [18]. Therefore, we predicted the secondary structure of the 50 -UTR of H-ras, K-Ras, and N-Ras using the MFOLD program [23]. The sequences of Hras, K-Ras, and N-Ras 50 -UTR were predicted to form several highly structured stem loops (data not shown). Similarly, Kumari et al. [24] reported that N-Ras contains a highly conserved, thermodynamically stable RNA G-quadruplex in the native 50 -UTR. Thus, we speculate that an alteration in eIF4E expression might preferentially affect the translationally weak Ras mRNA. Another possible explanation for the preferential reduction of Ras translation is that RCAN1 may be an mRNA binding protein. A stretch of roughly 80 amino acids near the N-terminus of this protein shows similarity with the RNA recognition motif, which could exert nucleic acid-binding functions [25]. Whether RCAN1-
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Fig. 5. RCAN1-4 knockdown decreases Ras translation. (A) Autoradiography (top) and densitometric (right) analysis showing newly synthesized Ras protein following [35S]methionine/cysteine labeling for the indicated periods from U87MG cells transfected with indicated siRNA. Coomassie blue staining of the SDS–PAGE gel as a loading control (bottom). (B) Autoradiography (top) and densitometric (right) analysis showing overall protein synthesis. Coomassie blue staining of the SDS–PAGE gel as a loading control (bottom). (C) Impaired assembly of the eIF4F complex in 293T cells transfected with siRCAN1-4. After pull-down, proteins eluted from m7GTP-sepharose 4B (upper) and whole cell lysates (lower) were analyzed by immunoblot.
4 binds with the mRNA of specific proteins and controls their translation is unknown, but this possibility is currently under investigation. The biological significance of cell growth control by RCAN1 has been demonstrated in physiologic angiogenesis, tumor angiogene-
sis, and its growth. Patients with Down syndrome have a different risk and clinical prognosis of certain malignancies. The complicated and diverse functions of RCAN1 on cell growth suggest that a pathway-specific approach is needed to target this gene for antiangiogenic and anticancer therapy.
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Fig. 6. siRCAN1-4 inhibits translation by a calcineurin-independent mechanism (A) Immunoblot showing that knocking down calcineurin A does not block siRCAN1-4induced reduction of Ras. U87MG cells were co-transfected with either siRNA targeting calcineurin Aa (siCnAa) or b (siCnAb), and either siRCAN1-4 or siCon in combinations as indicated. (B) Immunoblot showing that calcineurin inhibitors cyclosporine A (CsA; 1 lM) or FK506 (50 nM) does not block the reduced response to bFGF (10 ng/ml) stimulation in siRCAN1-4-transfected cells. (C–E) Immunoblot showing the effects of RCAN1 knockdown (C), calcineurin A knockdown (D), or the over-expression of calcineurin A mutants (E) on vector-mediated GFP expression. GFP transcript levels were shown by RT-PCR.
Acknowledgement This research was supported by a R13-2002-005-04002-0 from MRC for Cell Death Disease Research Center funded by Korea Science and Engineering Foundation (KOSEF).
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[8] Ermak, G., Harris, C.D. and Davies, K.J. (2002) The DSCR1 (Adapt78) isoform 1 protein calcipressin 1 inhibits calcineurin and protects against acute calciummediated stress damage, including transient oxidative stress. FASEB J. 16, 814– 824. [9] Qin, L., Zhao, D., Liu, X., Nagy, J.A., Hoang, M.V., Brown, L.F., Dvorak, H.F. and Zeng, H. (2006) Down syndrome candidate region 1 isoform 1 mediates angiogenesis through the calcineurin-NFAT pathway. Mol. Cancer Res. 4, 811– 820. [10] Zhang, F.L. and Casey, P.J. (1996) Protein prenylation: molecular mechanisms and functional consequences. Annu. Rev. Biochem. 65, 241–269. [11] Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X114 solution. J. Biol. Chem. 256, 1604–1607. [12] Yang, J., Rothermel, B., Vega, R.B., Frey, N., McKinsey, T.A., Olson, E.N., BasselDuby, R. and Williams, R.S. (2000) Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles. Circ. Res. 87, E61–E68. [13] Gray, N.K. and Wickens, M. (1998) Control of translation initiation in animals. Annu. Rev. Cell Dev. Biol. 14, 399–458. [14] Ryeom, S., Baek, K.H., Rioth, M.J., Lynch, R.C., Zaslavsky, A., Birsner, A., Yoon, S.S. and McKeon, F. (2008) Targeted deletion of the calcineurin inhibitor DSCR1 suppresses tumor growth. Cancer Cell 13, 420–431. [15] Kourtis, N. and Tavernarakis, N. (2009) Cell-specific monitoring of protein synthesis in vivo. PLoS ONE 4, e4547. [16] Saelens, X., Kalai, M. and Vandenabeele, P. (2001) Translation inhibition in apoptosis: caspase-dependent PKR activation and eIF2-alpha phosphorylation. J. Biol. Chem. 276, 41620–41628. [17] Iizuka, M., Abe, M., Shiiba, K., Sasaki, I. and Sato, Y. (2004) Down syndrome candidate region 1, a downstream target of VEGF, participates in endothelial cell migration and angiogenesis. J. Vasc. Res. 41, 334–344. [18] Dever, T.E. (2002) Gene-specific regulation by general translation factors. Cell 108, 545–556. [19] Rajasekhar, V.K., Viale, A., Socci, N.D., Wiedmann, M., Hu, X. and Holland, E.C. (2003) Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol. Cell 12, 889– 901. [20] Moerke, N.J. et al. (2007) Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. Cell 128, 257–267. [21] Hilioti, Z. et al. (2004) GSK-3 kinases enhance calcineurin signaling by phosphorylation of RCNs. Genes Dev. 18, 35–47. [22] Sans, M.D. and Williams, J.A. (2004) Calcineurin is required for translational control of protein synthesis in rat pancreatic acini. Am. J. Physiol. Cell Physiol. 287, C310–C319.
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RCAN1-4 knockdown attenuates cell growth through the inhibition of Ras signaling Hong Joon Lee a,b, Young Sun Kim a,b, Yasufumi Sato c, Young-Jin Cho a,b,* a b c
Department of Pharmacology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea Cell Death Disease Center of MRC, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea Department of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan
a r t i c l e
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Article history: Received 25 May 2009 Revised 12 June 2009 Accepted 11 July 2009 Available online 18 July 2009 Edited by Lukas Huber Keywords: RCAN1/DSCR1 Cell growth Ras ERK/MAPK Translation eIF4E
a b s t r a c t Forced changes in the expression of regulator of calcineurin 1 (RCAN1) affects cell growth. This has been linked to the suppression of calcineurin-nuclear factor of activated T cells signaling by RCAN1. Here, we describe a novel role of RCAN1 isoform 4 in proper expression of Ras protein and its signaling. RCAN1 isoform 4 knockdown attenuated growth factor-induced extracellular signal-regulated kinase activation and cell growth; reduced Ras levels and its translation rate; and led to a reduction of eukaryotic initiation factor 4E in the initiation complex and a slight repression of global protein synthesis. Experiments utilizing activity-modified mutants of calcineurin A demonstrated that these effects were calcineurin-independent. Our findings reveal a previously unknown role of RCAN1-4 in protein synthesis, which may be relevant to cell growth. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The human regulator of calcineurin 1 (RCAN1) gene, also termed Down syndrome candidate region 1, was first isolated near the portion of the Down Syndrome Critical Region on chromosome 21 [1]. The gene consists of seven exons; exons 1–4 can be alternatively spliced, resulting in four different transcripts denoted as RCAN1-1 through RCAN1-4. RCAN1 protein binds to the catalytic subunit of a Ca2+/calmodulin-dependent phosphatase calcineurin and inhibits its function [2]. Elevated RCAN1 expression has been observed in neurodegenerative diseases, including Alzheimer’s disease and Down syndrome [3], in the heart after pressure-overload [4], and in the peri-infarct cortex following experimental stroke [5]. Altered RCAN1 expression exerts diverse influences on cell proliferation. Over-expression of hamster homologue adapt78 induces G1-phase growth arrest in hemagglutinin (HA)-1 cells [6]. Consistently, adenovirus-mediated introduction of RCAN1-4 into endoAbbreviations: RCAN1, regulator of calcineurin 1; NFAT, nuclear factor of activated T cells; ERK, extracellular signal-regulated kinase; eIF4E, eukaryotic initiation factor 4E; GFP, green fluorescent protein; HA, hemagglutinin; GST, glutathione S-transferase * Corresponding authors. Address: Department of Pharmacology, College of Medicine, The Catholic University of Korea, Banpo-dong 505, Seocho-gu, Seoul 137701, Republic of Korea. Fax: +82 2 536 2485. E-mail address: [email protected] (Y.-J. Cho).
thelial cells induces G0/G1 arrest and inhibits angiogenesis [7]. On the other hand, the over-expression of RCAN1-1 promotes the proliferation of PC-12 cells [8]. In a study addressing isoform-specific functions, over-expression of RCAN1-1 induced endothelial cell proliferation, but RCAN1-1-specific knockdown did not affect the baseline cell growth. On the other hand, over-expression of RCAN1-4 did not affect baseline endothelial cell growth, and RCAN1-4-specific knockdown enhanced cell proliferation [9]. These observations suggest that RCAN1 may play differential roles in cell growth, dependent on cell types and isoforms of RCAN1. Until now, the speculated role of RCAN1 in cell growth has been mainly based on its function as a negative regulator of calcineurin signaling. Hence, the effects on growth-related signal transduction molecules have not been elucidated in detail. Here, we investigated the effects of altered RCAN1-4 expression on cell proliferation and growth factor-induced activation of extracellular signal-regulated kinase (ERK) pathway.
2. Materials and methods 2.1. Cell culture Human U87MG glioblastoma cells and human 293T embryonic kidney cancer cells were obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in minimal
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.07.023
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essential medium (U87) and Dulbecco’s modified Eagle’s medium (293T) supplemented with 1.0 mM sodium pyruvate and 10% fetal bovine serum under an atmosphere of 5% CO2 at 37 °C. If needed, the cell number was counted using a Z2 Coulter Counter (Multisizer II, Beckman–Coulter). 2.2. Biochemical procedures Chemicals and antibodies, plasmid construction, short interfering RNA (siRNA)-mediated RNA interference, metabolic cell labeling and immunoprecipitation, pulse-chase analysis, pull-down assays, luciferase assay, Triton X- 114 separation, real-time quantitative RT-PCR analysis, and immunoblot analysis were performed according to standard protocols. Details can be found in the Supplementary data. 3. Results 3.1. RCAN1-4 knockdown suppresses U87MG cell growth We compared the growth rate of U87MG cells with RCAN1-4 knocked down and control cells. To suppress the expression of endogenous RCAN1-4, we designed siRNA targeted against exon 4 (siRCAN1-4). Transfection with this siRNA resulted in an approximate 90% reduction in mRNA levels, which were quantified by real-time RT-PCR analysis (Fig. 1A). After transfection with siRCAN1-4 or control siRNA, the number of cells in culture was counted every 24 h until 96 h. The cell numbers for siRCAN1-4transfected and control cultures were not significantly different 24 and 48 h after the transfection. However, at 72 h and 96 h, the growth of siRCAN1-4-transfected cells was significantly retarded relative to control cells (29% and 35%, respectively; Fig. 1B). Earlier reports demonstrated the arrest of cell cycle progression in RCAN1modulated cells [6,7]. To investigate the involvement of cyclindependent kinase inhibitors in the retardation of cell growth, we examined the level of p21 and p27Kip1. As shown in Fig. 1C, siRCAN1-4 significantly increased p21 and p27 Kip1 expression in a time-dependent manner. 3.2. RCAN1-4 knockdown attenuates the growth factor-induced ERK activation Next, we examined ERK activation in response to growth factors. Compared with controls, siRCAN1-4-transfected cells exhibited an attenuated induction of ERK phosphorylation following bFGF treatment (Fig. 2A). Attenuated ERK phosphorylation was also observed in response to phorbol 12-myristate 13-acetate and calcium ionophore A23187 stimulation (data not shown). We examined the activation of the upstream ERK signal to identify a target molecule that is directly inhibited by RCAN1-4 knockdown. Transfection with siRCAN1-4 attenuated the EGF-induced phosphorylation of Raf-1 and MEK (Fig. 2B). Notably, the level of Ras protein was also reduced in siRCAN1-4-transfected cells relative to controls. Transcription of the reporter gene mediated by Elk-1, a downstream effector molecule of ERK, was also reduced in siRCAN1-4-transfected cells (Fig. 2C). Reciprocally, vector-mediated over-expression of green fluorescent protein (GFP)-RCAN1-4 partially rescued the attenuated phosphorylation of ERK in siRCAN14-transfected cells (Fig. 2D). 3.3. RCAN1-4 knockdown reduces steady-state levels of Ras protein Based on the reduction of Ras expression, we hypothesized that the attenuated response of the ERK pathway to growth fac-
Fig. 1. RCAN1 knockdown suppresses U87MG cell growth. (A) Real-time PCR for RCAN1-4 (left) and detection of RCAN1-4 expression (right) in U87MG cells transfected with either siRCAN1-4 or siCon. (B) U87MG cells were transfected with either a specific siRNA for RCAN1 isoform 4 (siRCAN1-4) or control siRNA (siCon), and cell growth was determined by a Coulter counter. Results are the means and S.D. of three independent experiments (*P < 0.05, **P < 0.001 versus time-matched control). (C) Representative immunoblot showing the time-dependent change of p21 and p27Kip1 expressions following transfection with either siRCAN1-4 or siCon.
tors resulted from a deficit of Ras-mediated signal transduction. We examined Ras activation following the stimulation of EGF. As expected, the amount of GTP-bound Ras in siRCAN1-4-transfected cells was significantly low compared to control (by 42% and 31% at 10 min and 30 min after stimulation, respectively; Fig. 3A). We then investigated whether there is a change in the synthesis of Ras and its post-translational prenylation, which is important for Ras protein localization to the plasma membrane and receiving signals from receptors [10]. The cytosolic and membrane-bound forms of Ras were separated by Triton X-114 and quantified by Western blot analysis [11]. Prenylated Ras protein in detergent phase was reduced by 47% in siRCAN1-4-transfected cells compared to control (Fig. 3B). A longer exposure of
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Fig. 2. RCAN1-4 knockdown attenuates growth factor-induced ERK activation. (A) Time course study showing bFGF (10 ng/ml)-induced ERK phosphorylation (top) in U87MG cells transfected with either siRCAN1-4 or siCon. Densitometric ratios of the p-ERK/ERK signal (bottom) are represented as mean and S.D. from three independent immunoblots (*P < 0.05 versus time-matched control). (B) Immunoblot showing EGF (20 ng/ml for 10 min) induced phosphorylation of signaling molecules in an upstream ERK pathway of 293T cells. (C) Attenuation of Elk-1 promoter-driven luciferase expression following EGF treatment (20 ng/ml for 4 h) in siRCAN1-4-transfected 293T cells. Results are the means and S.D. of four independent experiments. *P < 0.05 compared with siCon. (D) Immunoblot of EGF (20 ng/ml for 10 min) induced phosphorylation of ERK in 293T cells co-transfected with either a vector encoding RCAN1-4 (GFP-RCAN1) or GFP, and either siRCAN1-4 or siCon in the combinations indicated.
the same panel showed that non-prenylated Ras in aqueous phase was also reduced by 45%. These observations suggest that siRCAN1-4 down-regulates the overall level of Ras protein expression rather than a post-translational maturation process. Consistent with the ERK response to EGF (Fig. 2C), over-expression of GFP-RCAN1-4 partially rescued Ras expression (Fig. 3C). We next examined an isoform-specific effect of RCAN1. Cells were transfected with siRCAN1-4 and siRCAN1-1 specifically targeted to exon1. Transfection of siRCAN1-1 inhibited Ras expression by 17% (Fig. 3D). Interestingly, transfection of siRCAN1-1 increased the transcription of endogenous RCAN1-4 by twofold (data not shown). The transcription of RCAN1-4 gene is activated potently by calcineurin due to the presence of multiple consensus nuclear factor of activated T cells (NFAT) binding sites within an internal promoter region [12]. Thus this increase of RCAN1-4 transcript is probably due to the release of calcineurin from an inhibitory effect of RCAN1-1. In the next experiments, we used only RCAN1-4.
3.4. RCAN1-4 knockdown does not affect Ras transcription and protein stability The cellular protein level is the net result of protein synthesis and degradation. To explore the molecular basis of the reduction in Ras expression, we first examined transcriptional level of Ras isoforms. Real-time PCR analysis revealed that H- and K-Ras mRNA levels were not significantly decreased, and N-Ras transcription was rather increased in siRCAN1-4-transfected cells (Fig. 4A). These results suggest that reduced expression of Ras protein was not due to suppression in transcriptional stage. To investigate the stability of Ras protein, we next examined whether it is actively degraded by various cellular proteases. siRCAN1-4-transfected or control cells were treated for 8 h with the proteasomal inhibitor MG132 and the lysosomotrophic agent chloroquine. The cells were then subjected to immunoblot analysis for Ras. Inhibition of these proteases did not block the reduction of Ras expression in siRCAN1-4-transfected cells (Fig. 4B). To confirm that siRCAN1-4 does
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Fig. 3. RCAN1-4 knockdown reduces the steady-state level of Ras protein. (A) Time course study showing the formation of GTP-bound Ras (GTP-Ras) after EGF treatment (20 ng/ml) to 293T cells transfected with indicated siRNA, which was assayed by pull-down (PD) with glutathione S-transferase-RBD. (B) Immunoblot showing the attenuated expression of prenylated (upper bands) and non-prenylated Ras (lower bands) in siRCAN1-4-transfected 293T cells. a, aqueous phase; d, Triton X-114 detergent phase containing lipid-associated proteins. The relative density of bands was given below. The value obtained in siCon-transfected cells were set as 100% (C) Immunoblot showing that co-transfection of GFP-RCAN1-4 increases Ras expression in 293T cells transfected with siRCAN1-4. (D) Immunoblot showing effects of isoform-specific siRCAN1s on Ras expression in U87MG cells.
not affect Ras protein stability, we performed a pulse-chase analysis for Ras degradation. The cells were pulsed with [35S]-methionine/cysteine for 4 h and then chased for 20 h. Quantitative analysis of the remaining radiolabeled Ras revealed that the degradation rates of Ras protein were not significantly different between the siRCAN1-4-transfected cells and controls (Fig. 4C). Collectively, these results indicate that the reduction in Ras expression is not due to enhanced protein degradation or attenuated transcription.
(eIF4E-BP1) levels. To determine whether these alterations could disrupt the eIF4E complex, we examined the association of eIFs by pulling down with m7GTP-sepharose resin. The precipitation assay showed a reduced association of eIF4E and eIF4E-BP1, whereas the amounts of eIF4A and eIF4G were not significantly altered (Fig. 5C). These findings suggest an inhibitory role for RCAN1-4 in the translation initiation processes, probably by down-regulating eIF4E.
3.5. RCAN1-4 knockdown decreases Ras protein translation
3.6. siRCAN1-4 inhibits translation by a calcineurin-independent mechanism
We next examined the translational efficiency of Ras protein. The cells were cultured in a medium containing [35S]-methionine/cysteine to radiolabel the newly synthesized proteins for two time periods: 30 and 60 min. After the indicated incubation, cell lysates were obtained and analyzed by SDS–PAGE. Quantitative imaging with a phosphorimager demonstrated that newly synthesized Ras in siRCAN1-4-transfected cells was 64.6% and 74% less than that of time-matched controls at 30 and 60 min, respectively (Fig. 5A). Overall, 35S-labeled cellular protein was also reduced in RCAN1-4-transfected cells by about 20% relative to control cells (Fig. 5B). It is notable that Ras synthesis is more intensively inhibited than global translation. These observations suggest a role for RCAN1-4 in translational regulation. The eukaryotic initiation factors eIF4E, eIF4G, and eIF4A comprise the trimeric eIF4F complex, which promotes cap-dependent mRNA translation [13]. Therefore, we tested whether RCAN1-4 knockdown alters the expression of eIFs or assembly of the eIF4F complex. Transfection with siRCAN1-4 had no measurable effect on the levels of eIF4A and eIF4G proteins but resulted in reduced eIF4E and eIF4E-binding protein
RCAN1 is an endogenous inhibitor of calcineurin, and targeted deletion or knockdown of this gene results in the activation of calcineurin [9,14]. We next examined whether the elevated calcineurin activity in siRCAN1-4-transfected cells contributes to the attenuated translation of Ras protein. We knocked down either calcineurin Aa (siCnAa) or calcineurin Ab (siCnAb) simultaneously with RCAN1-4. Reducing the abundance of calcineurin did not rescue Ras expression in siRCAN1-4-transfected cells (Fig. 6A). Similarly, pharmacological inhibitors of calcineurin, cyclosporine A and FK506, did not restore the attenuated ERK response to bFGF (Fig. 6B). We then investigated whether the translation deficit in siRCAN1-4-transfected cells was also independent of calcineurin. We examined the translational efficiency in siCnA-transfected cells and siRCAN1-4-transfected cells by monitoring vector-mediated expression of GFP as a reporter [15,16]. The GFP expression vector was transfected 24 h after siRNA transfection, and cells were incubated another 24 h before immunoblot analysis. Consistent with the results of a metabolic labeling experiment for the analysis of
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Fig. 4. RCAN1-4 knockdown does not affect Ras transcription and protein stability. (A) Effects of siRCAN1-4 on the Ras transcript levels of 293T cells measured by realtime RT-PCR. *P < 0.05 compared with siCon. (B) Immunoblot showing that a proteasome inhibitor (MG132; 4 lM for 8 h) or lysosome inhibitor (chloroquine; 200 lV for 8 h) does not block the siRCAN1-4-induced reduction of Ras in U87MG cells. (C) Degradation of Ras protein. U87MG cells were pulsed with [35S]methionine/cysteine for 4 h. Radiolabeled Ras was chased for 20 h.
protein synthesis, the transfection of siRCAN1-4 significantly inhibited the expression of GFP (Fig. 6C). Interestingly, the siCnAs also inhibited GFP expression instead of an expected opposite effect to that of siRCAN1-4 (Fig. 6D). Next, we assessed whether calcineurin activation enhanced Ras and GFP expression. Transfection of the constitutively active form of calcineurin increased Ras and GFP expression by 10% and 32%, respectively, indicating a positive role of calcineurin A in Ras and GFP translation (Fig. 6E). Taken together, these data suggest that siRCAN does not inhibit Ras translation by augmenting calcineurin signaling. 4. Discussion The evidence that proper RCAN1 expression plays an important role in proliferation has been observed in diverse cell type. RCAN1 is involved in proliferation as well as growth suppression [6– 9,14,17]. Furthermore, each RCAN1 isoform plays differential roles in cell proliferation in response to a growth factor [9]. Thus, the
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mechanism by which RCAN1 modulates cell proliferation is complicated and not completely understood. In contrast with the previous report utilizing vascular endothelial cells [9], we demonstrated that RCAN1-4 knockdown results in the retarded growth of U87MG cells and the attenuated activation of the ERK pathway in response to growth factors. In addition, our results showed for the first time that RCAN1-4 knockdown suppressed translation initiation complex formation and total protein synthesis in a calcineurin-independent manner. This may affect cell growth because many growth regulators are encoded by weak mRNAs, translation of which is more sensitive to small perturbations in translation initiation complex formation [13,18–20]. Among them, this study showed Ras protein as one of the signaling molecules relevant to cell growth and sensitive to the defect of translation induced by knockdown of RCAN1-4. RCAN1-4 reportedly works as a negative regulator of cell growth by inhibiting calcineurin/NFAT pathway-dependent mechanism [7,9]. Thus, RCAN1-4 knockdown can enhance cell growth via a calcineurin-dependent mechanism [9], and can also suppress cell growth via a calcineurin-independent mechanism, which is suggested by this study. The dominance between these opposite events may be affected by the temporal status and types of cells. In addition to this, isoform-specific differential effects of RCAN1 on calcineurin may explain the diversity of growth responses to RCAN1 modulation. In addition, the phosphorylation status of RCAN1 is reported to affect calcineurin signaling [21]. Thus, the overall effects of RCAN1 on growth would be extremely diverse depending on the cellular context. The independence of siRCAN1-4-induced down-regulation of Ras on the calcineurin pathway was shown in several experiments utilizing pharmacological inhibitors, siRNAs, and calcineurin A mutants. Although a mechanism was not addressed in this study, these experiments revealed that calcineurin is a positive regulator of Ras translation in U87MG cells. Similarly, a requirement for calcineurin in translational machinery activation has been reported in rat pancreatic acini [22]. Given that RCAN1-4 is a negative regulator of calcineurin, RCAN1-4 knockdown is expected to exert influence on Ras expression into two modes: facilitation of translation by activating a calcineurin-dependent mechanism and translation inhibition via a calcineurin-independent mechanism. The mechanism by which RCAN1-4 knockdown preferentially inhibits the translation of Ras protein is not completely solved in this study. In this respect, we showed that knocking down RCAN1-4 reduced eIF4E expression and its binding to the 7methyl-GTP cap structure. In eukaryotes, roughly 90% of protein is synthesized through cap-dependent translation. Indeed, knocking down RCAN1-4 reduced global translation in U87MG cells. This observation raised the question of why Ras expression was strongly inhibited compared to that of other proteins observed in this study. Cellular mRNAs differ hugely in the amount of eIF4F complex required for efficient translation [13]. The translation efficiency of mRNA with highly complex 50 -untranslated regions (50 UTR) is especially dependent on eIF4E levels [18]. Therefore, we predicted the secondary structure of the 50 -UTR of H-ras, K-Ras, and N-Ras using the MFOLD program [23]. The sequences of Hras, K-Ras, and N-Ras 50 -UTR were predicted to form several highly structured stem loops (data not shown). Similarly, Kumari et al. [24] reported that N-Ras contains a highly conserved, thermodynamically stable RNA G-quadruplex in the native 50 -UTR. Thus, we speculate that an alteration in eIF4E expression might preferentially affect the translationally weak Ras mRNA. Another possible explanation for the preferential reduction of Ras translation is that RCAN1 may be an mRNA binding protein. A stretch of roughly 80 amino acids near the N-terminus of this protein shows similarity with the RNA recognition motif, which could exert nucleic acid-binding functions [25]. Whether RCAN1-
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Fig. 5. RCAN1-4 knockdown decreases Ras translation. (A) Autoradiography (top) and densitometric (right) analysis showing newly synthesized Ras protein following [35S]methionine/cysteine labeling for the indicated periods from U87MG cells transfected with indicated siRNA. Coomassie blue staining of the SDS–PAGE gel as a loading control (bottom). (B) Autoradiography (top) and densitometric (right) analysis showing overall protein synthesis. Coomassie blue staining of the SDS–PAGE gel as a loading control (bottom). (C) Impaired assembly of the eIF4F complex in 293T cells transfected with siRCAN1-4. After pull-down, proteins eluted from m7GTP-sepharose 4B (upper) and whole cell lysates (lower) were analyzed by immunoblot.
4 binds with the mRNA of specific proteins and controls their translation is unknown, but this possibility is currently under investigation. The biological significance of cell growth control by RCAN1 has been demonstrated in physiologic angiogenesis, tumor angiogene-
sis, and its growth. Patients with Down syndrome have a different risk and clinical prognosis of certain malignancies. The complicated and diverse functions of RCAN1 on cell growth suggest that a pathway-specific approach is needed to target this gene for antiangiogenic and anticancer therapy.
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Fig. 6. siRCAN1-4 inhibits translation by a calcineurin-independent mechanism (A) Immunoblot showing that knocking down calcineurin A does not block siRCAN1-4induced reduction of Ras. U87MG cells were co-transfected with either siRNA targeting calcineurin Aa (siCnAa) or b (siCnAb), and either siRCAN1-4 or siCon in combinations as indicated. (B) Immunoblot showing that calcineurin inhibitors cyclosporine A (CsA; 1 lM) or FK506 (50 nM) does not block the reduced response to bFGF (10 ng/ml) stimulation in siRCAN1-4-transfected cells. (C–E) Immunoblot showing the effects of RCAN1 knockdown (C), calcineurin A knockdown (D), or the over-expression of calcineurin A mutants (E) on vector-mediated GFP expression. GFP transcript levels were shown by RT-PCR.
Acknowledgement This research was supported by a R13-2002-005-04002-0 from MRC for Cell Death Disease Research Center funded by Korea Science and Engineering Foundation (KOSEF).
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