- Email: [email protected]
Crosstalk between ERK2 and RXR regulates nuclear import of transcription factor NGFI-B
BBRC Biochemical and Biophysical Research Communications 336 (2005) 646–652 www.elsevier.com/locate/ybbrc
Crosstalk between ERK2 and RXR regulates nuclear import of transcription factor NGFI-B q Chris M. Jacobs, Ragnhild E. Paulsen * Department of Pharmaceutical Biosciences, University of Oslo, Norway Received 12 August 2005 Available online 29 August 2005
Abstract Transcription factor NGFI-B initiates apoptosis when allowed to translocate to mitochondria. Retinoid-X receptor (RXR), another member of the nuclear receptor family, regulates NGFI-B signaling through heterodimerization and nuclear export. Growth factor EGF activates ERK2, which phosphorylates NGFI-B and determines if NGFI-B is allowed to translocate to mitochondria. In the present study, EGF treatment resulted in an increased nuclear import of NGFI-B. Likewise, active ERK2 resulted in a preferential nuclear localization of NGFI-B. When coexpressed with RXR the nuclear import and nuclear localization induced by active ERK2 were strongly reduced. In the presence of its ligand 9-cis-retinoic acid, RXR no longer inhibited ERK2-induced nuclear import. Thus, RXR serves a permissive role for ERK2-mediated nuclear accumulation of NGFI-B. This finding represents a novel crosstalk between ERK2 and RXR signaling pathways, and explains how two independent inhibitors of apoptosis (EGF and 9-cis-retinoic acid) may cooperate to regulate nuclear targeting of apoptosis inducer NGFI-B. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Apoptosis; 9-cis-Retinoic acid; EGF; ERK2; FRAP; NGFI-B; Nuclear import; RXR; Transcription factor
Transcription factor NGFI-B, also called Nur77 or TR3, is an immediate early gene and an orphan member of the nuclear receptor family (the steroid–thyroid receptor superfamily) [1]. NGFI-B was originally identified because of its rapid induction by serum in fibroblasts and by NGF in PC12 pheochromocytoma cells [2,3]. NGFI-B is called an orphan nuclear receptor due to its strong sequence homology to nuclear receptors, however, no specific ligand has yet been identified [4]. Although NGFI-B is transcribed at a low level in many tissues and shows developmental changes [5], it may be further activated transiently, rapidly, and independent of protein synthesis in a variety of cell
q Abbreviations: EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; NGFI-B, NGF-induced clone B, RXR, retinoid-X receptor; 9cRA, 9-cis retinoic acid. * Corresponding author. Fax: +47 22 84 49 44. E-mail address: [email protected] (R.E. Paulsen).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.08.143
types by multiple stimuli, including growth factors [6,7]. The importance of NGFI-B in the apoptotic process was originally determined in knock-out experiments in T-cell hybridomas [8,9]. During apoptosis, the NGFI-B protein has a function distinct from that of a transcription factor; it translocates to mitochondria to initiate the apoptotic process [10]. It has been demonstrated that NGFI-B interacts with Bcl-2, thereby converting Bcl-2 from a protector to killer [11]. Thus, the subcellular localization of NGFI-B is important for its biological effects, since it functions in the nucleus to induce gene regulation and proliferation, and on the mitochondria to induce apoptosis. Therefore, NGFI-B is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization [12]. The subcellular localization of NGFI-B is tightly regulated, the mechanisms of which are yet only partly understood. Retinoid-X receptor alpha (RXRa) is another member of the nuclear receptor family [13]. RXRs mediate gene regulation through RXR/RXR homodimers, RXR/retinoic
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
acid receptor heterodimers [13] as well as through heterodimerization with several other nuclear receptors including NGFI-B [14]. A high affinity ligand for RXRs, 9-cis-retinoic acid (9cRA) [15], induces transactivation by RXR homodimers as well as RXR/NGFI-B heterodimers [14]. In addition to its effect on gene activation, the ligand is important for nuclear export of NGFI-B. Specifically, RXRa ligands suppress a nuclear export sequence (NES) activity present in the carboxyl terminus of RXRa, by inducing RXRa homdimerization or altering RXRa/NGFI-B heterodimerization [16]. Consequently, RXR ligands were shown to inhibit mitochondrial targeting of RXRa/ NGFI-B heterodimers as well as their ability to induce apoptosis [16]. Epidermal growth factor (EGF) as another cell survival factor is widely known to initiate the activation of the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. The importance of the ERK pathway in cell survival has been well documented in PC12 cells where activation of the ERK signaling pathway inhibits apoptosis, whereas the down-regulation of ERK mediated by neuronal growth factor (NGF) withdrawal leads to apoptosis [17]. EGF stimulation has been shown to lead to phosphorylation of NGFI-B in PC12 cells [6]. In addition, NGFI-B is a direct substrate for the MAP kinase ERK2 [18] and is prohibited from apoptosis-induced mitochondrial targeting in the presence of active ERK2 [19]. The objective of the present study was therefore to investigate the potential crosstalk between ERK2 and RXR signaling pathways, and to explain how independent inhibitors of apoptosis may cooperate to regulate the nuclear targeting of apoptosis inducer NGFI-B. Materials and methods DulbeccoÕs MEM with 25 mM Hepes (DMEM) and fetal bovine serum were obtained from Gibco (Paisley, Scotland). Luciferin was from Duchefa Biochemie BV (Haarlem, The Netherlands). Recombinant human EGF was obtained from PeproTech (NJ, USA). All other reagents were from Sigma. Transient transfection of CV1 cells. CV1 cells were grown in DulbeccoÕs modified EagleÕs medium supplemented with 10% fetal bovine serum. A calcium phosphate precipitation procedure was used for transient transfections. Expressor plasmids encoding wild type NGFI-B or gfp-tagged NGFI-B have been described earlier [18,19]. The ERK2 plasmids pCMV5 ERK2-MEK1R4F (dominant active) and pCMV5 ERK2 K52R (dominant negative) were kindly provided by Dr. M. Cobb, University of Texas, South Western Medical Center, Dallas, USA, and pCMX/hRXRa was a gift from Dr. R. Evans (The Salk Institute, San Diego, USA). The NGFIB reporter plasmid was NBRE8-LUC [4]. Each transfection reaction was carried out with a total of 1 lg DNA/ml medium. For luciferase assay CV1 cells were transfected with 0.1 lg NBRE8-LUC, 0,1 lg of wild type NGFI-B, 0,4 lg of the different ERK2 plasmids or RXR and filled up to a total of 1 lg DNA/ml medium by pCMV (empty expressor plasmid). For FRAP experiments wild type NGFI-B plasmid was replaced by gfp-tagged NGFI-B [19]. After transfection, the cells were incubated further for 20– 24 h and then EGF (5 ng/ml) or 9cRA (10 lM) was added directly into the medium. Luciferase activity was measured following 6 h of incubation with ligands, as earlier described [20], using an EG&G Berthold Lumat LB9507 luminometer.
647
FRAP analysis. CV1 cells were plated in LabTek II chambers (Nalge Nunc International, USA) and selective photobleaching of the nucleus was performed using a 488 nm laser on a Nikon TE2000-E microscope configured with a C1 Nikon confocal unit run by EZ-C1 software. Laser was set to 6 amps and 100% power for 15 s for bleaching the nucleus. Fluorescence recovery was monitored by scanning the whole cell every minute for 15 min with the laser set to 3 amps and 85% power. Gain was set to 5.5 for all experiments.
Results NGFI-B expressed in CV-1 cells redistributes to the nucleus when given EGF or coexpressed with active ERK2 (ERK2MEK1R4F) or ligand-bound RXR To find the subcellular localization of NGFI-B following treatment with the antiapoptotic growth factor EGF or RXR ligand 9cRA, we transfected CV-1 cells with gfptagged NGFI-B alone or together with active ERK2 (ERK2-MEK1R4F) or dominant negative ERK2 (ERK2K52R) and/or RXR, and treated with EGF (5 ng/ml) or 9cRA (10 lM). The cells were investigated using confocal microscopy (Fig. 1). In untreated cells, NGFI-B distributed evenly between the nucleus and cytoplasm. EGF treatment resulted in a transiently increased nuclear accumulation of NGFI-B (Fig. 1 and [19]). Consistently, coexpression of ERK2-MEK1R4F resulted in a sustained and predominant nuclear localization of NGFI-B. When gfp-tagged NGFI-B was coexpressed with RXR in the absence of the ligand 9cRA, no qualitative difference was seen when comparing to NGFI-B alone. When adding 9cRA, an increased and long-lasting nuclear localization was seen; however, it was less prominent than with ERK2MEK1R4F (Fig. 1). Since both active ERK2 and ligandbound RXR affected the subcellular distribution of NGFI-B, we investigated the crosstalk between the two pathways by coexpressing ERK2-MEK1R4F and RXR together with gfp-tagged NGFI-B. In the absence of 9cRA RXR suppressed the ERK2-induced nuclear accumulation of NGFI-B. Only when 9cRA was present NGFI-B was allowed to accumulate in the nucleus (Fig. 1). Active ERK2 or RXR in the presence of 9cRA increases transcriptional activity by NGFI-B, whereas RXR in the absence of 9cRA suppresses ERK2-induced NGFI-B transcriptional activity Since the nuclear localization of gfp-tagged NGFI-B was highly dependent on ERK2 and RXR, it was interesting to investigate if there was a cross-talk between these two signaling pathways to the transcriptional activity of wild type NGFI-B (no gfp tag), as measured by the activity of an NGFI-B-responsive reporter gene expressing luciferase. EGF treatment did not increase the expression of luciferase (Fig. 2A), consistent with the transient nuclear accumulation of gfp-tagged NGFI-B. However, when ERK2 was allowed to be constitutively active with the cotransfection with plasmid ERK2-MEK1R4F, an
648
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
A
300
*
250 200 150 100 50 0
NGFI-B EGF MEK1R4F K52R
B
-
+ -
+ -
+ -
+ + -
+ + -
+ +
300 # *
250 #
200 150 100 50 0
9cRA NGFI-B RXR MEK1R4F K52R
-
+ -
+ -
+ + -
+ -
+ + -
+ + -
+ + + -
+ + + -
+ + + + -
+ + +
+ + + +
Fig. 2. Active ERK2 or RXR in the presence of 9cRA increases transcriptional activity by NGFI-B, whereas RXR in the absence of 9cRA suppresses ERK2-induced NGFI-B transcriptional activity. CV1 cells were transfected with NGFI-B alone or together with ERK2MEK1R4F or ERK2-K52R (A) or together with RXR and ERK2MEK1R4F or REK2-K52R (B). EGF (5 ng/ml) or 9cRA (10 lM) was given directly into the medium and the luciferase activity was measured following 6 h of incubation. Luciferase activity values are given as percent of NGFI-B alone and are means ± SE from 5 to 11 observations from 5 independent experiments. *p < 0.05 comparing to NGFI-B alone (one-way ANOVA followed by DunnÕs post hoc test); #p < 0.05 comparing the presence of 9cRA to absence (sign test). Fig. 1. Green fluorescence protein (gfp)-tagged NGFI-B expressed in CV1 cells redistributes when given EGF or coexpressed with active ERK2 (ERK2-MEK1R4F) or RXR. CV1 cells were transfected with gfp-tagged NGFI-B alone or together with ERK2-MEK1R4F and/or RXR. EGF (5 ng/ml) or 9cRA (10 lM) was given directly into the medium and the cells were observed at 3–4 min following treatment. The cells were analyzed with a C1 Nikon confocal unit.
increased luciferase activity was observed. Such increase in reporter gene activity was not observed when the cells were cotransfected with dominant negative ERK2 (ERK2K52R) (Fig. 2A). When RXR was present together with wild type NGFI-B in the transfected cells, the addition of 9cRA resulted in an increased transcription of the reporter gene, consistent with the role of RXR as a coactivator for NGFI-B transcriptional activity (Fig. 2B). Together with RXR, a cotransfection of ERK2-MEK1R4F with NGFIB did not result in increased transcription of the reporter gene compared to NGFI-B alone, except when 9cRA was present (Fig. 2B). Thus, RXR serves a permissive role for ERK2 regulation of NGFI-B nuclear accumulation (Fig. 1) and hence transcriptional activity (Fig. 2).
EGF treatment increases the rate of nuclear import of NGFI-B It was known that RXR may influence NGFI-B nuclear localization by RXR-induced nuclear export of NGFI-B. This export is inhibited in the presence of RXR ligand, which suppresses NES activity of RXR [16]. The mechanism of EGF or ERK2-induced nuclear accumulation was unknown. We have therefore analyzed fluorescence recovery after photobleaching (FRAP) in cells transfected with gfp-tagged NGFI-B in the absence or presence of EGF (Fig. 3A). We have investigated a 15-min time period following photobleaching and observed a continuous fluorescence recovery leveling off at the end of this period (Fig. 3B). By measuring the slope of the first minute of recovery it was possible to quantify the nuclear import of NGFI-B (Fig. 3C). Nuclear export did not contribute to this value, since pretreatment by nuclear export inhibitor leptomycin B (10 ng/ml) as in [21] did not increase this (not shown). When EGF was added, there was an increased
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
649
Fig. 3. EGF treatment increases the rate of nuclear import of NGFI-B. Quantitative nuclear FRAP analysis of gfp-tagged NGFI-B in cells treated with EGF (5 ng/ml). Images were obtained before photobleaching and at the indicated timepoints thereafter. The nucleus of individual cells was photobleached (A). The average nuclear fluorescence recovery (B) and the slope of the initial 1 min recovery were calculated (C). Values are means from 3 or 7 individual cells.
rate of accumulation of NGFI-B compared to untreated cells. Thus, EGF induces an increased nuclear localization of NGFI-B (Fig. 1) by an effect on nuclear import. Non-liganded RXR decreases the rate of nuclear import of NGFI-B Since RXR reduced ERK2-induced NGFI-B nuclear localization (Fig. 1) and transcriptional activity (Fig. 2), it was interesting to investigate its effect on nuclear import of NGFI-B in the absence or presence of ligand (9cRA) (Fig. 4). Gfp-tagged NGFI-B was photobleached in cells cotransfected with RXR (Fig. 4A). The presence of RXR reduced the rate of recovery after photobleaching compared to cells expressing NGFI-B alone (Figs. 4B and C). In the presence of 9cRA, however, RXR no longer inhibited nuclear import of NGFI-B (Fig. 4C).
RXR prohibits ERK2-induced nuclear arrest of NGFI-B Since both EGF and RXR influenced nuclear import, it was interesting to investigate crosstalk between ERK2 and RXR. We have therefore photobleached cells cotransfected with gfp-labeled NGFI-B and active ERK2 (ERK2MEK1R4F) in the absence or presence of RXR (Fig. 5). When NGFI-B was expressed in the presence of active ERK, NGFI-B was mainly localized to the nucleus (Fig. 5A). The very small amount of remaining cytoplasmic gfp-tagged NGFI-B after photobleaching was imported slowly into the nucleus (Figs. 5B and C). However, when RXR was present, there was no prebleach nuclear arrest of NGFI-B. Furthermore, the slope of recovery was higher than with NGFI-B alone, probably due to the presence of active ERK2, but lower than NGFI-B with added EGF (Fig. 5 compared to Fig. 3), consistent with an inhibition
650
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
Fig. 4. Non-liganded RXR decreases the rate of nuclear import of NGFI-B. Quantitative nuclear FRAP analysis of gfp-tagged NGFI-B in cells coexpressing RXR in the presence or absence of 9cRA (10 lM). Images were obtained before photobleaching and at the indicated timepoints thereafter. The nucleus of individual cells was photobleached (A). The average nuclear fluorescence recovery (B) and the slope of the initial 1 min recovery were calculated (C). Values are means from 4 to 7 individual cells.
of ERK-2 mediated increased nuclear import by non-liganded RXR. When adding 9cRA, the slope of recovery increased only slightly further (if at all) (Figs. 5B and C), consistent with a main effect of ligand-bound RXR on inhibition of nuclear export rather than nuclear import. Discussion In this communication, we show that EGF treatment or active ERK2 results in an increased nuclear import of NGFI-B and that RXRa serves a permissive role for ERK2-mediated nuclear accumulation of NGFI-B. NGFI-B was evenly distributed between the nucleus and cytosol (Fig. 1). It functions in the nucleus as a transcription factor to regulate gene expression by binding to specific response elements using a well-conserved DNA-binding
domain [4]. Its role as a transcription factor is regulated by heterodimerization with other members in the nuclear receptor family, such as RXR [14]. Heterodimerization of RXR with NGFI-B enhances DNA binding and subsequently transcriptional activation [14] as confirmed in Fig. 2. DNA binding and transactivation are required for induction of cell proliferation in lung cancer cells [12]. More recent studies have shown that NGFI-B may also act outside the nucleus to induce apoptosis or differentiation. In response to apoptotic stimuli, NGFI-B targets mitochondria and converts Bcl-2 from a protector to a killer by direct protein interaction [11]. This translocation of NGFI-B to mitochondria requires the heterodimerization with RXRa, where RXRa serves as a shuttling molecule in the NGFI-B-dependent apoptosis and NGFI-B is the executor [16]. In response to nerve growth factor (NGF)
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
651
Fig. 5. RXR prohibits ERK2-induced nuclear arrest of NGFI-B. Quantitative nuclear FRAP analysis of gfp-tagged NGFI-B in cells coexpressing RXR and/or ERK2-MEK1R4F in the presence or absence of 9cRA (10 lM). Images were obtained before photobleaching and at the indicated timepoints thereafter. The nucleus of individual cells or left cell was photobleached (A). The average nuclear fluorescence recovery (B) and the slope of the initial 1 min recovery were calculated (C). Values are means from 4 to 7 individual cells.
treatment NGFI-B translocated from the nucleus to the cytosol in PC12 cells, suggesting that cytoplasmic action of NGFI-B was required for NGF-induced PC12 cell differentiation [21]. Thus, the subcellular localization of NGFI-B is critical for its biological effects. The NGFI-B protein contains nuclear localization signals (NLSs) as well as several nuclear export signals (NESs). NGFI-B has three NESs which are critical for RXRa/NGFI-B nuclear export [21]. In addition, RXRa contains an NES, which contributes to efficient nuclear export of the RXRa/NGFI-B heterodimer [16]. This RXRa NES may be active in the
absence of ligand and is silenced when 9cRA is present. During apoptosis, RXRa and NGFI-B preferentially dimerize through their DNA-binding domain interfaces to activate RXRa NES, resulting in their cytoplasmic localization [16]. It is presently unknown how an apoptotic stimulus activates RXRa NES [16]. Binding by RXR ligands may induce a dimerization interface switch to the carboxyl-terminus ligand binding domain, that silences the RXRa NES, leading to RXRa/NGFI-B nuclear localization and efficient transcriptional regulation [16]. RXR ligands have earlier been shown to have an inhibitory effect
652
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
on apoptosis [22], and a mechanism is now suggested to be an inhibition of RXRa/NGFI-B mitochondrial targeting [16]. The data in the present communication are in agreement with these mechanisms, since NGFI-B coexpressed with RXRa was cytoplasmic in the absence of 9cRA, and had increased nuclear localization in the presence of 9cRA (Fig. 1), as well as inhibition of nuclear import in the absence of 9cRA (Fig. 4). RXRa exists as a monomer in the cytoplasm, but in response to 9cRA resides as a homodimer in the nucleus [16]. Thus, the presence of cytoplasmic RXRa in the absence of ligand slows down the nuclear import of NGFI-B, whereas nuclear ligand-bound RXRa slows down nuclear export of NGFI-B, rather than affecting nuclear import (Figs. 4 and 5). Another signaling pathway which may protect from apoptosis is EGF treatment. Protection seen by EGF treatment is mainly gene regulated in some cells, whereas other cells show the presence of a protective mechanism which is rapid and independent of prior gene regulation [19]. In this latter case, EGF treatment led to a transient nuclear accumulation of NGFI-B which was dependent on ERK2 activity [19]. In the present communication, we show that EGF treatment results in an increased nuclear import of NGFIB, since the slope of the initial fluorescence recovery after photobleaching was increased (Fig. 3). Thus, nuclear export as well as nuclear import of NGFI-B may be targeted when protecting against NGFI-B-dependent apoptosis. Since both RXRa and EGF affected nuclear targeting of NGFI-B, it was interesting to investigate the crosstalk between the two pathways. When RXRa and ERK2 were coexpressed in the cells, RXRa served a permissive role for the ERK2-induced nuclear targeting of NGFI-B, such that RXRa in the absence of ligand strongly reduced the nuclear accumulation of NGFI-B. Thus, cells expressing high levels of RXRa may be more resistant to EGF-induced protection against NGFI-B-dependent apoptosis in the absence of ligand. In conclusion, these results show a novel crosstalk between ERK2 and RXRa signaling pathways, and explain how two independent inhibitors of apoptosis (EGF and 9-cis-retinoic acid) may cooperate to regulate nuclear targeting of apoptosis inducer NGFI-B. Acknowledgments The authors thank Mona Gaarder for expert technical assistance. Financial support from the Norwegian Research Council (Grant NFR147574/310) and Center for Cellular Stress Responses (Medical Faculty of the University of Oslo) is gratefully acknowledged. References [1] R.M. Evans, The steroid and thyroid hormone receptor superfamily, Science 240 (1988) 889–895. [2] J. Milbrandt, Nerve growth factor induces a gene homologous to the glucocorticoid receptor gene, Neuron 1 (1988) 183–188.
[3] T.G. Hazel, D. Nathans, L.F. Lau, A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily, Proc. Natl. Acad. Sci. USA 85 (1988) 8444–8448. [4] T.E. Wilson, T.J. Fahrner, M. Johnston, J. Milbrandt, Identification of the DNA binding site for NGFI-B by genetic selection in yeast, Science 252 (1991) 1296–1300. [5] M.A. Watson, J. Milbrandt, Expression of the nerve growth factorregulated NGFI-A and NGFI-B genes in the developing rat, Development 110 (1990) 173–183. [6] T.G. Hazel, R. Misra, I.J. Davis, M.E. Greenberg, L.F. Lau, Nur77 is differentially modified in PC12 cells upon membrane depolarization and growth factor treatment, Mol. Cell. Biol. 11 (1991) 3239–3246. [7] E. Ciani, R.E. Paulsen, Activation of a reporter gene responsive to NGFI-B in cultured neurons and astrocytes, J. Mol. Neurosci. 6 (1995) 131–139. [8] J.D. Woronicz, B. Calnan, V. Ngo, A. Winoto, Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas, Nature 367 (1994) 277–281. [9] Z.G. Liu, S.W. Smith, K.A. McLaughlin, L.M. Schwartz, B.A. Osborne, Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77, Nature 367 (1994) 281–284. [10] H. Li, S.K. Kolluri, J. Gu, M.I. Dawson, X. Cao, P.D. Hobbs, B. Lin, G. Chen, J. Lu, F. Lin, Z. Xie, J.A. Fontana, J.C. Reed, X. Zhang, Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3, Science 289 (2000) 1159– 1164. [11] B. Lin, S.K. Kolluri, F. Lin, W. Liu, Y.H. Han, X. Cao, M.I. Dawson, J.C. Reed, X.K. Zhang, Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3, Cell 116 (2004) 527–540. [12] S.K. Kolluri, N. Bruey-Sedano, X. Cao, B. Lin, F. Lin, Y.H. Han, M.I. Dawson, X.K. Zhang, Mitogenic effect of orphan receptor TR3 and its regulation by MEKK1 in lung cancer cells, Mol. Cell. Biol. 23 (2003) 8651–8667. [13] D.J. Mangelsdorf, R.M. Evans, The RXR heterodimers and orphan receptors, Cell 83 (1995) 841–850. [14] T. Perlmann, L. Jansson, A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1, Genes Dev. 9 (1995) 769–782. [15] R.A. Heyman, D.J. Mangelsdorf, J.A. Dyck, R.B. Stein, G. Eichele, R.M. Evans, C. Thaller, 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor, Cell 68 (1992) 397–406. [16] X. Cao, W. Liu, F. Lin, H. Li, S.K. Kolluri, B. Lin, Y.H. Han, M.I. Dawson, X.K. Zhang, Retinoid X receptor regulates Nur77/TR3dependent apoptosis [corrected] by modulating its nuclear export and mitochondrial targeting, Mol. Cell. Biol. 24 (2004) 9705–9725. [17] Z. Xia, M. Dickens, J. Raingeaud, R.J. Davis, M.E. Greenberg, Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis, Science 270 (1995) 1326–1331. [18] H.H. Slagsvold, A.C. Ostvold, A.B. Fallgren, R.E. Paulsen, Nuclear receptor and apoptosis initiator NGFI-B is a substrate for kinase ERK2, Biochem. Biophys. Res. Commun. 291 (2002) 1146–1150. [19] C.M. Jacobs, K.A. Boldingh, H.H. Slagsvold, G.H. Thoresen, R.E. Paulsen, ERK2 prohibits apoptosis-induced subcellular translocation of orphan nuclear receptor NGFI-B/TR3, J. Biol. Chem. 279 (2004) 50097–50101. [20] R.E. Paulsen, C.A. Weaver, T.J. Fahrner, J. Milbrandt, Domains regulating transcriptional activity of the inducible orphan receptor NGFI-B, J. Biol. Chem. 267 (1992) 16491–16496. [21] Y. Katagiri, K. Takeda, Z.X. Yu, V.J. Ferrans, K. Ozato, G. Guroff, Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B, Nat. Cell Biol. 2 (2000) 435–440. [22] R.P. Bissonnette, R.T. Brunner, S.B. Lazarchik, N.J. Yoo, M.F. Boehm, D.R. Green, R.A. Heyman, 9-cis retinoic acid inhibition of activation-induced apoptosis is mediated via regulation of fas ligand and requires retinoic acid receptor and retinoid X receptor activation, Mol. Cell. Biol. 15 (1995) 5576–5585.
Crosstalk between ERK2 and RXR regulates nuclear import of transcription factor NGFI-B q Chris M. Jacobs, Ragnhild E. Paulsen * Department of Pharmaceutical Biosciences, University of Oslo, Norway Received 12 August 2005 Available online 29 August 2005
Abstract Transcription factor NGFI-B initiates apoptosis when allowed to translocate to mitochondria. Retinoid-X receptor (RXR), another member of the nuclear receptor family, regulates NGFI-B signaling through heterodimerization and nuclear export. Growth factor EGF activates ERK2, which phosphorylates NGFI-B and determines if NGFI-B is allowed to translocate to mitochondria. In the present study, EGF treatment resulted in an increased nuclear import of NGFI-B. Likewise, active ERK2 resulted in a preferential nuclear localization of NGFI-B. When coexpressed with RXR the nuclear import and nuclear localization induced by active ERK2 were strongly reduced. In the presence of its ligand 9-cis-retinoic acid, RXR no longer inhibited ERK2-induced nuclear import. Thus, RXR serves a permissive role for ERK2-mediated nuclear accumulation of NGFI-B. This finding represents a novel crosstalk between ERK2 and RXR signaling pathways, and explains how two independent inhibitors of apoptosis (EGF and 9-cis-retinoic acid) may cooperate to regulate nuclear targeting of apoptosis inducer NGFI-B. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Apoptosis; 9-cis-Retinoic acid; EGF; ERK2; FRAP; NGFI-B; Nuclear import; RXR; Transcription factor
Transcription factor NGFI-B, also called Nur77 or TR3, is an immediate early gene and an orphan member of the nuclear receptor family (the steroid–thyroid receptor superfamily) [1]. NGFI-B was originally identified because of its rapid induction by serum in fibroblasts and by NGF in PC12 pheochromocytoma cells [2,3]. NGFI-B is called an orphan nuclear receptor due to its strong sequence homology to nuclear receptors, however, no specific ligand has yet been identified [4]. Although NGFI-B is transcribed at a low level in many tissues and shows developmental changes [5], it may be further activated transiently, rapidly, and independent of protein synthesis in a variety of cell
q Abbreviations: EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; NGFI-B, NGF-induced clone B, RXR, retinoid-X receptor; 9cRA, 9-cis retinoic acid. * Corresponding author. Fax: +47 22 84 49 44. E-mail address: [email protected] (R.E. Paulsen).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.08.143
types by multiple stimuli, including growth factors [6,7]. The importance of NGFI-B in the apoptotic process was originally determined in knock-out experiments in T-cell hybridomas [8,9]. During apoptosis, the NGFI-B protein has a function distinct from that of a transcription factor; it translocates to mitochondria to initiate the apoptotic process [10]. It has been demonstrated that NGFI-B interacts with Bcl-2, thereby converting Bcl-2 from a protector to killer [11]. Thus, the subcellular localization of NGFI-B is important for its biological effects, since it functions in the nucleus to induce gene regulation and proliferation, and on the mitochondria to induce apoptosis. Therefore, NGFI-B is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization [12]. The subcellular localization of NGFI-B is tightly regulated, the mechanisms of which are yet only partly understood. Retinoid-X receptor alpha (RXRa) is another member of the nuclear receptor family [13]. RXRs mediate gene regulation through RXR/RXR homodimers, RXR/retinoic
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
acid receptor heterodimers [13] as well as through heterodimerization with several other nuclear receptors including NGFI-B [14]. A high affinity ligand for RXRs, 9-cis-retinoic acid (9cRA) [15], induces transactivation by RXR homodimers as well as RXR/NGFI-B heterodimers [14]. In addition to its effect on gene activation, the ligand is important for nuclear export of NGFI-B. Specifically, RXRa ligands suppress a nuclear export sequence (NES) activity present in the carboxyl terminus of RXRa, by inducing RXRa homdimerization or altering RXRa/NGFI-B heterodimerization [16]. Consequently, RXR ligands were shown to inhibit mitochondrial targeting of RXRa/ NGFI-B heterodimers as well as their ability to induce apoptosis [16]. Epidermal growth factor (EGF) as another cell survival factor is widely known to initiate the activation of the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. The importance of the ERK pathway in cell survival has been well documented in PC12 cells where activation of the ERK signaling pathway inhibits apoptosis, whereas the down-regulation of ERK mediated by neuronal growth factor (NGF) withdrawal leads to apoptosis [17]. EGF stimulation has been shown to lead to phosphorylation of NGFI-B in PC12 cells [6]. In addition, NGFI-B is a direct substrate for the MAP kinase ERK2 [18] and is prohibited from apoptosis-induced mitochondrial targeting in the presence of active ERK2 [19]. The objective of the present study was therefore to investigate the potential crosstalk between ERK2 and RXR signaling pathways, and to explain how independent inhibitors of apoptosis may cooperate to regulate the nuclear targeting of apoptosis inducer NGFI-B. Materials and methods DulbeccoÕs MEM with 25 mM Hepes (DMEM) and fetal bovine serum were obtained from Gibco (Paisley, Scotland). Luciferin was from Duchefa Biochemie BV (Haarlem, The Netherlands). Recombinant human EGF was obtained from PeproTech (NJ, USA). All other reagents were from Sigma. Transient transfection of CV1 cells. CV1 cells were grown in DulbeccoÕs modified EagleÕs medium supplemented with 10% fetal bovine serum. A calcium phosphate precipitation procedure was used for transient transfections. Expressor plasmids encoding wild type NGFI-B or gfp-tagged NGFI-B have been described earlier [18,19]. The ERK2 plasmids pCMV5 ERK2-MEK1R4F (dominant active) and pCMV5 ERK2 K52R (dominant negative) were kindly provided by Dr. M. Cobb, University of Texas, South Western Medical Center, Dallas, USA, and pCMX/hRXRa was a gift from Dr. R. Evans (The Salk Institute, San Diego, USA). The NGFIB reporter plasmid was NBRE8-LUC [4]. Each transfection reaction was carried out with a total of 1 lg DNA/ml medium. For luciferase assay CV1 cells were transfected with 0.1 lg NBRE8-LUC, 0,1 lg of wild type NGFI-B, 0,4 lg of the different ERK2 plasmids or RXR and filled up to a total of 1 lg DNA/ml medium by pCMV (empty expressor plasmid). For FRAP experiments wild type NGFI-B plasmid was replaced by gfp-tagged NGFI-B [19]. After transfection, the cells were incubated further for 20– 24 h and then EGF (5 ng/ml) or 9cRA (10 lM) was added directly into the medium. Luciferase activity was measured following 6 h of incubation with ligands, as earlier described [20], using an EG&G Berthold Lumat LB9507 luminometer.
647
FRAP analysis. CV1 cells were plated in LabTek II chambers (Nalge Nunc International, USA) and selective photobleaching of the nucleus was performed using a 488 nm laser on a Nikon TE2000-E microscope configured with a C1 Nikon confocal unit run by EZ-C1 software. Laser was set to 6 amps and 100% power for 15 s for bleaching the nucleus. Fluorescence recovery was monitored by scanning the whole cell every minute for 15 min with the laser set to 3 amps and 85% power. Gain was set to 5.5 for all experiments.
Results NGFI-B expressed in CV-1 cells redistributes to the nucleus when given EGF or coexpressed with active ERK2 (ERK2MEK1R4F) or ligand-bound RXR To find the subcellular localization of NGFI-B following treatment with the antiapoptotic growth factor EGF or RXR ligand 9cRA, we transfected CV-1 cells with gfptagged NGFI-B alone or together with active ERK2 (ERK2-MEK1R4F) or dominant negative ERK2 (ERK2K52R) and/or RXR, and treated with EGF (5 ng/ml) or 9cRA (10 lM). The cells were investigated using confocal microscopy (Fig. 1). In untreated cells, NGFI-B distributed evenly between the nucleus and cytoplasm. EGF treatment resulted in a transiently increased nuclear accumulation of NGFI-B (Fig. 1 and [19]). Consistently, coexpression of ERK2-MEK1R4F resulted in a sustained and predominant nuclear localization of NGFI-B. When gfp-tagged NGFI-B was coexpressed with RXR in the absence of the ligand 9cRA, no qualitative difference was seen when comparing to NGFI-B alone. When adding 9cRA, an increased and long-lasting nuclear localization was seen; however, it was less prominent than with ERK2MEK1R4F (Fig. 1). Since both active ERK2 and ligandbound RXR affected the subcellular distribution of NGFI-B, we investigated the crosstalk between the two pathways by coexpressing ERK2-MEK1R4F and RXR together with gfp-tagged NGFI-B. In the absence of 9cRA RXR suppressed the ERK2-induced nuclear accumulation of NGFI-B. Only when 9cRA was present NGFI-B was allowed to accumulate in the nucleus (Fig. 1). Active ERK2 or RXR in the presence of 9cRA increases transcriptional activity by NGFI-B, whereas RXR in the absence of 9cRA suppresses ERK2-induced NGFI-B transcriptional activity Since the nuclear localization of gfp-tagged NGFI-B was highly dependent on ERK2 and RXR, it was interesting to investigate if there was a cross-talk between these two signaling pathways to the transcriptional activity of wild type NGFI-B (no gfp tag), as measured by the activity of an NGFI-B-responsive reporter gene expressing luciferase. EGF treatment did not increase the expression of luciferase (Fig. 2A), consistent with the transient nuclear accumulation of gfp-tagged NGFI-B. However, when ERK2 was allowed to be constitutively active with the cotransfection with plasmid ERK2-MEK1R4F, an
648
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
A
300
*
250 200 150 100 50 0
NGFI-B EGF MEK1R4F K52R
B
-
+ -
+ -
+ -
+ + -
+ + -
+ +
300 # *
250 #
200 150 100 50 0
9cRA NGFI-B RXR MEK1R4F K52R
-
+ -
+ -
+ + -
+ -
+ + -
+ + -
+ + + -
+ + + -
+ + + + -
+ + +
+ + + +
Fig. 2. Active ERK2 or RXR in the presence of 9cRA increases transcriptional activity by NGFI-B, whereas RXR in the absence of 9cRA suppresses ERK2-induced NGFI-B transcriptional activity. CV1 cells were transfected with NGFI-B alone or together with ERK2MEK1R4F or ERK2-K52R (A) or together with RXR and ERK2MEK1R4F or REK2-K52R (B). EGF (5 ng/ml) or 9cRA (10 lM) was given directly into the medium and the luciferase activity was measured following 6 h of incubation. Luciferase activity values are given as percent of NGFI-B alone and are means ± SE from 5 to 11 observations from 5 independent experiments. *p < 0.05 comparing to NGFI-B alone (one-way ANOVA followed by DunnÕs post hoc test); #p < 0.05 comparing the presence of 9cRA to absence (sign test). Fig. 1. Green fluorescence protein (gfp)-tagged NGFI-B expressed in CV1 cells redistributes when given EGF or coexpressed with active ERK2 (ERK2-MEK1R4F) or RXR. CV1 cells were transfected with gfp-tagged NGFI-B alone or together with ERK2-MEK1R4F and/or RXR. EGF (5 ng/ml) or 9cRA (10 lM) was given directly into the medium and the cells were observed at 3–4 min following treatment. The cells were analyzed with a C1 Nikon confocal unit.
increased luciferase activity was observed. Such increase in reporter gene activity was not observed when the cells were cotransfected with dominant negative ERK2 (ERK2K52R) (Fig. 2A). When RXR was present together with wild type NGFI-B in the transfected cells, the addition of 9cRA resulted in an increased transcription of the reporter gene, consistent with the role of RXR as a coactivator for NGFI-B transcriptional activity (Fig. 2B). Together with RXR, a cotransfection of ERK2-MEK1R4F with NGFIB did not result in increased transcription of the reporter gene compared to NGFI-B alone, except when 9cRA was present (Fig. 2B). Thus, RXR serves a permissive role for ERK2 regulation of NGFI-B nuclear accumulation (Fig. 1) and hence transcriptional activity (Fig. 2).
EGF treatment increases the rate of nuclear import of NGFI-B It was known that RXR may influence NGFI-B nuclear localization by RXR-induced nuclear export of NGFI-B. This export is inhibited in the presence of RXR ligand, which suppresses NES activity of RXR [16]. The mechanism of EGF or ERK2-induced nuclear accumulation was unknown. We have therefore analyzed fluorescence recovery after photobleaching (FRAP) in cells transfected with gfp-tagged NGFI-B in the absence or presence of EGF (Fig. 3A). We have investigated a 15-min time period following photobleaching and observed a continuous fluorescence recovery leveling off at the end of this period (Fig. 3B). By measuring the slope of the first minute of recovery it was possible to quantify the nuclear import of NGFI-B (Fig. 3C). Nuclear export did not contribute to this value, since pretreatment by nuclear export inhibitor leptomycin B (10 ng/ml) as in [21] did not increase this (not shown). When EGF was added, there was an increased
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
649
Fig. 3. EGF treatment increases the rate of nuclear import of NGFI-B. Quantitative nuclear FRAP analysis of gfp-tagged NGFI-B in cells treated with EGF (5 ng/ml). Images were obtained before photobleaching and at the indicated timepoints thereafter. The nucleus of individual cells was photobleached (A). The average nuclear fluorescence recovery (B) and the slope of the initial 1 min recovery were calculated (C). Values are means from 3 or 7 individual cells.
rate of accumulation of NGFI-B compared to untreated cells. Thus, EGF induces an increased nuclear localization of NGFI-B (Fig. 1) by an effect on nuclear import. Non-liganded RXR decreases the rate of nuclear import of NGFI-B Since RXR reduced ERK2-induced NGFI-B nuclear localization (Fig. 1) and transcriptional activity (Fig. 2), it was interesting to investigate its effect on nuclear import of NGFI-B in the absence or presence of ligand (9cRA) (Fig. 4). Gfp-tagged NGFI-B was photobleached in cells cotransfected with RXR (Fig. 4A). The presence of RXR reduced the rate of recovery after photobleaching compared to cells expressing NGFI-B alone (Figs. 4B and C). In the presence of 9cRA, however, RXR no longer inhibited nuclear import of NGFI-B (Fig. 4C).
RXR prohibits ERK2-induced nuclear arrest of NGFI-B Since both EGF and RXR influenced nuclear import, it was interesting to investigate crosstalk between ERK2 and RXR. We have therefore photobleached cells cotransfected with gfp-labeled NGFI-B and active ERK2 (ERK2MEK1R4F) in the absence or presence of RXR (Fig. 5). When NGFI-B was expressed in the presence of active ERK, NGFI-B was mainly localized to the nucleus (Fig. 5A). The very small amount of remaining cytoplasmic gfp-tagged NGFI-B after photobleaching was imported slowly into the nucleus (Figs. 5B and C). However, when RXR was present, there was no prebleach nuclear arrest of NGFI-B. Furthermore, the slope of recovery was higher than with NGFI-B alone, probably due to the presence of active ERK2, but lower than NGFI-B with added EGF (Fig. 5 compared to Fig. 3), consistent with an inhibition
650
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
Fig. 4. Non-liganded RXR decreases the rate of nuclear import of NGFI-B. Quantitative nuclear FRAP analysis of gfp-tagged NGFI-B in cells coexpressing RXR in the presence or absence of 9cRA (10 lM). Images were obtained before photobleaching and at the indicated timepoints thereafter. The nucleus of individual cells was photobleached (A). The average nuclear fluorescence recovery (B) and the slope of the initial 1 min recovery were calculated (C). Values are means from 4 to 7 individual cells.
of ERK-2 mediated increased nuclear import by non-liganded RXR. When adding 9cRA, the slope of recovery increased only slightly further (if at all) (Figs. 5B and C), consistent with a main effect of ligand-bound RXR on inhibition of nuclear export rather than nuclear import. Discussion In this communication, we show that EGF treatment or active ERK2 results in an increased nuclear import of NGFI-B and that RXRa serves a permissive role for ERK2-mediated nuclear accumulation of NGFI-B. NGFI-B was evenly distributed between the nucleus and cytosol (Fig. 1). It functions in the nucleus as a transcription factor to regulate gene expression by binding to specific response elements using a well-conserved DNA-binding
domain [4]. Its role as a transcription factor is regulated by heterodimerization with other members in the nuclear receptor family, such as RXR [14]. Heterodimerization of RXR with NGFI-B enhances DNA binding and subsequently transcriptional activation [14] as confirmed in Fig. 2. DNA binding and transactivation are required for induction of cell proliferation in lung cancer cells [12]. More recent studies have shown that NGFI-B may also act outside the nucleus to induce apoptosis or differentiation. In response to apoptotic stimuli, NGFI-B targets mitochondria and converts Bcl-2 from a protector to a killer by direct protein interaction [11]. This translocation of NGFI-B to mitochondria requires the heterodimerization with RXRa, where RXRa serves as a shuttling molecule in the NGFI-B-dependent apoptosis and NGFI-B is the executor [16]. In response to nerve growth factor (NGF)
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
651
Fig. 5. RXR prohibits ERK2-induced nuclear arrest of NGFI-B. Quantitative nuclear FRAP analysis of gfp-tagged NGFI-B in cells coexpressing RXR and/or ERK2-MEK1R4F in the presence or absence of 9cRA (10 lM). Images were obtained before photobleaching and at the indicated timepoints thereafter. The nucleus of individual cells or left cell was photobleached (A). The average nuclear fluorescence recovery (B) and the slope of the initial 1 min recovery were calculated (C). Values are means from 4 to 7 individual cells.
treatment NGFI-B translocated from the nucleus to the cytosol in PC12 cells, suggesting that cytoplasmic action of NGFI-B was required for NGF-induced PC12 cell differentiation [21]. Thus, the subcellular localization of NGFI-B is critical for its biological effects. The NGFI-B protein contains nuclear localization signals (NLSs) as well as several nuclear export signals (NESs). NGFI-B has three NESs which are critical for RXRa/NGFI-B nuclear export [21]. In addition, RXRa contains an NES, which contributes to efficient nuclear export of the RXRa/NGFI-B heterodimer [16]. This RXRa NES may be active in the
absence of ligand and is silenced when 9cRA is present. During apoptosis, RXRa and NGFI-B preferentially dimerize through their DNA-binding domain interfaces to activate RXRa NES, resulting in their cytoplasmic localization [16]. It is presently unknown how an apoptotic stimulus activates RXRa NES [16]. Binding by RXR ligands may induce a dimerization interface switch to the carboxyl-terminus ligand binding domain, that silences the RXRa NES, leading to RXRa/NGFI-B nuclear localization and efficient transcriptional regulation [16]. RXR ligands have earlier been shown to have an inhibitory effect
652
C.M. Jacobs, R.E. Paulsen / Biochemical and Biophysical Research Communications 336 (2005) 646–652
on apoptosis [22], and a mechanism is now suggested to be an inhibition of RXRa/NGFI-B mitochondrial targeting [16]. The data in the present communication are in agreement with these mechanisms, since NGFI-B coexpressed with RXRa was cytoplasmic in the absence of 9cRA, and had increased nuclear localization in the presence of 9cRA (Fig. 1), as well as inhibition of nuclear import in the absence of 9cRA (Fig. 4). RXRa exists as a monomer in the cytoplasm, but in response to 9cRA resides as a homodimer in the nucleus [16]. Thus, the presence of cytoplasmic RXRa in the absence of ligand slows down the nuclear import of NGFI-B, whereas nuclear ligand-bound RXRa slows down nuclear export of NGFI-B, rather than affecting nuclear import (Figs. 4 and 5). Another signaling pathway which may protect from apoptosis is EGF treatment. Protection seen by EGF treatment is mainly gene regulated in some cells, whereas other cells show the presence of a protective mechanism which is rapid and independent of prior gene regulation [19]. In this latter case, EGF treatment led to a transient nuclear accumulation of NGFI-B which was dependent on ERK2 activity [19]. In the present communication, we show that EGF treatment results in an increased nuclear import of NGFIB, since the slope of the initial fluorescence recovery after photobleaching was increased (Fig. 3). Thus, nuclear export as well as nuclear import of NGFI-B may be targeted when protecting against NGFI-B-dependent apoptosis. Since both RXRa and EGF affected nuclear targeting of NGFI-B, it was interesting to investigate the crosstalk between the two pathways. When RXRa and ERK2 were coexpressed in the cells, RXRa served a permissive role for the ERK2-induced nuclear targeting of NGFI-B, such that RXRa in the absence of ligand strongly reduced the nuclear accumulation of NGFI-B. Thus, cells expressing high levels of RXRa may be more resistant to EGF-induced protection against NGFI-B-dependent apoptosis in the absence of ligand. In conclusion, these results show a novel crosstalk between ERK2 and RXRa signaling pathways, and explain how two independent inhibitors of apoptosis (EGF and 9-cis-retinoic acid) may cooperate to regulate nuclear targeting of apoptosis inducer NGFI-B. Acknowledgments The authors thank Mona Gaarder for expert technical assistance. Financial support from the Norwegian Research Council (Grant NFR147574/310) and Center for Cellular Stress Responses (Medical Faculty of the University of Oslo) is gratefully acknowledged. References [1] R.M. Evans, The steroid and thyroid hormone receptor superfamily, Science 240 (1988) 889–895. [2] J. Milbrandt, Nerve growth factor induces a gene homologous to the glucocorticoid receptor gene, Neuron 1 (1988) 183–188.
[3] T.G. Hazel, D. Nathans, L.F. Lau, A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily, Proc. Natl. Acad. Sci. USA 85 (1988) 8444–8448. [4] T.E. Wilson, T.J. Fahrner, M. Johnston, J. Milbrandt, Identification of the DNA binding site for NGFI-B by genetic selection in yeast, Science 252 (1991) 1296–1300. [5] M.A. Watson, J. Milbrandt, Expression of the nerve growth factorregulated NGFI-A and NGFI-B genes in the developing rat, Development 110 (1990) 173–183. [6] T.G. Hazel, R. Misra, I.J. Davis, M.E. Greenberg, L.F. Lau, Nur77 is differentially modified in PC12 cells upon membrane depolarization and growth factor treatment, Mol. Cell. Biol. 11 (1991) 3239–3246. [7] E. Ciani, R.E. Paulsen, Activation of a reporter gene responsive to NGFI-B in cultured neurons and astrocytes, J. Mol. Neurosci. 6 (1995) 131–139. [8] J.D. Woronicz, B. Calnan, V. Ngo, A. Winoto, Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas, Nature 367 (1994) 277–281. [9] Z.G. Liu, S.W. Smith, K.A. McLaughlin, L.M. Schwartz, B.A. Osborne, Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77, Nature 367 (1994) 281–284. [10] H. Li, S.K. Kolluri, J. Gu, M.I. Dawson, X. Cao, P.D. Hobbs, B. Lin, G. Chen, J. Lu, F. Lin, Z. Xie, J.A. Fontana, J.C. Reed, X. Zhang, Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3, Science 289 (2000) 1159– 1164. [11] B. Lin, S.K. Kolluri, F. Lin, W. Liu, Y.H. Han, X. Cao, M.I. Dawson, J.C. Reed, X.K. Zhang, Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3, Cell 116 (2004) 527–540. [12] S.K. Kolluri, N. Bruey-Sedano, X. Cao, B. Lin, F. Lin, Y.H. Han, M.I. Dawson, X.K. Zhang, Mitogenic effect of orphan receptor TR3 and its regulation by MEKK1 in lung cancer cells, Mol. Cell. Biol. 23 (2003) 8651–8667. [13] D.J. Mangelsdorf, R.M. Evans, The RXR heterodimers and orphan receptors, Cell 83 (1995) 841–850. [14] T. Perlmann, L. Jansson, A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1, Genes Dev. 9 (1995) 769–782. [15] R.A. Heyman, D.J. Mangelsdorf, J.A. Dyck, R.B. Stein, G. Eichele, R.M. Evans, C. Thaller, 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor, Cell 68 (1992) 397–406. [16] X. Cao, W. Liu, F. Lin, H. Li, S.K. Kolluri, B. Lin, Y.H. Han, M.I. Dawson, X.K. Zhang, Retinoid X receptor regulates Nur77/TR3dependent apoptosis [corrected] by modulating its nuclear export and mitochondrial targeting, Mol. Cell. Biol. 24 (2004) 9705–9725. [17] Z. Xia, M. Dickens, J. Raingeaud, R.J. Davis, M.E. Greenberg, Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis, Science 270 (1995) 1326–1331. [18] H.H. Slagsvold, A.C. Ostvold, A.B. Fallgren, R.E. Paulsen, Nuclear receptor and apoptosis initiator NGFI-B is a substrate for kinase ERK2, Biochem. Biophys. Res. Commun. 291 (2002) 1146–1150. [19] C.M. Jacobs, K.A. Boldingh, H.H. Slagsvold, G.H. Thoresen, R.E. Paulsen, ERK2 prohibits apoptosis-induced subcellular translocation of orphan nuclear receptor NGFI-B/TR3, J. Biol. Chem. 279 (2004) 50097–50101. [20] R.E. Paulsen, C.A. Weaver, T.J. Fahrner, J. Milbrandt, Domains regulating transcriptional activity of the inducible orphan receptor NGFI-B, J. Biol. Chem. 267 (1992) 16491–16496. [21] Y. Katagiri, K. Takeda, Z.X. Yu, V.J. Ferrans, K. Ozato, G. Guroff, Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B, Nat. Cell Biol. 2 (2000) 435–440. [22] R.P. Bissonnette, R.T. Brunner, S.B. Lazarchik, N.J. Yoo, M.F. Boehm, D.R. Green, R.A. Heyman, 9-cis retinoic acid inhibition of activation-induced apoptosis is mediated via regulation of fas ligand and requires retinoic acid receptor and retinoid X receptor activation, Mol. Cell. Biol. 15 (1995) 5576–5585.