JunD Mediates Survival Signaling by the JNK Signal Transduction Pathway

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1 Molecular Cell, Vol. 11, , June, 2003, Copyright 2003 by Cell Press JunD Mediates Survival Signaling by the JNK Signal Transduction Pathway Jennifer A. Lamb, 1 Juan-Jose Ventura, 1 Patricia Hess, 1 Richard A. Flavell, 2 and Roger J. Davis 1, * 1 Howard Hughes Medical Institute and Program in Molecular Medicine Department of Biochemistry & Molecular Biology University of Massachusetts Medical School Worcester, Massachusetts Howard Hughes Medical Institute and Section of Immunobiology Yale University School of Medicine New Haven, Connecticut Summary The c-jun NH 2 -terminal kinase (JNK) can cause cell death by activating the mitochondrial apoptosis pathway. However, JNK is also capable of signaling cell survival. The mechanism that accounts for the dual role of JNK in apoptosis and survival signaling has not been established. Here we demonstrate that JNK- stimulated survival signaling can be mediated by JunD. The JNK/JunD pathway can collaborate with NF- B to increase antiapoptotic gene expression. This observation accounts for the ability of JNK to cause either survival or apoptosis in different cellular contexts. Fur- thermore, these data illustrate the general principal that signal transduction pathway integration is critical for the ability of cells to mount an appropriate biologi- cal response to a specific challenge. Introduction Many signaling pathways can cause markedly different cellular responses, and the biological outcome can be dependent upon the specific cellular context. Examples include the TGF and ERK pathways that can cause either growth arrest or increased cellular proliferation. The mechanism that enables the same signaling path- way to mediate such different responses is unclear. Nevertheless, this plasticity of signal transduction pathway function appears to be critical for the ability of cells to mount an appropriate biological response to a specific stimulus. To investigate the mechanism of signaling plasticity, we have examined the c-jun NH 2 -terminal kinase (JNK) pathway, which can contribute to either apoptosis or survival responses. The JNK pathway has been implicated in stressinduced apoptosis (Davis, 2000). Gene disruption studies demonstrate that ablation of the JNK signaling pathway causes decreased apoptosis in response to environmental stress (Tournier et al., 2000). Furthermore, studies using constitutively activated Jnk alleles demonstrate that the JNK signaling pathway can cause apoptosis (Lei et al., 2002). The mechanism of JNK-stimulated apoptosis involves the mitochondrial pathway (Tournier et al., *Correspondence: roger.davis@umassmed.edu 2000) and requires the proapoptotic Bcl2-related proteins Bax and Bak (Lei et al., 2002). Although substantial evidence supports a functional role for the JNK signaling pathway in stress-stimulated apoptosis, some studies have indicated that JNK may also contribute to cell survival (Davis, 2000). Two examples of survival signaling by JNK have been defined by analysis of JNK-deficient mice. First, Jnk1 / Jnk2 / embryos exhibit markedly increased apoptosis in the forebrain (Kuan et al., 1999; Sabapathy et al., 1999). Second, JNK-deficient mice are resistant to Bcr/Abl-induced lymphoma because the transformed JNK-deficient B cells exhibit dramatically increased apoptosis (Hess et al., 2002). A role for JNK in cell survival signaling has also been established in studies using antisense oligonucleotides to downregulate JNK expression in some cultured cells (Potapova et al., 2000). Together, these data indicate that JNK can signal both cell survival and apoptosis. The dual role of JNK in both apoptotic and antiapo- ptotic signaling pathways indicates that the function of JNK is complex and that the physiological response most likely reflects a balance between the ability of JNK to signal both apoptosis and cell survival. The complex nature of the cellular response to JNK activation is reflected by the actions of tumor necrosis factor (TNF). This cytokine is an important regulator of immune re- sponses that also regulates cell differentiation, survival, and apoptosis. Binding of ligand to TNF receptor-1 (TNF-R1) causes trimerization of the receptor and the recruitment of the adaptor protein TRADD, which serves as a platform for the subsequent recruitment of FADD, TRAF2, and RIP1 (Chen and Goeddel, 2002). Apoptosis and anti-inflammatory responses to TNF are mediated by the activation of caspase-8 by FADD (Varfolomeev et al., 1998; Yeh et al., 1998). In contrast, anti-apoptosis and inflammatory responses are mediated by activation of the NF- B pathway by TRAF2 and RIP1 (Devin et al., 2000; Kelliher et al., 1998). The role of JNK in the re- sponse of cells to TNF is unclear. Previous studies have demonstrated that TNF increases JNK activity (Kyriakis et al., 1994; Sluss et al., 1994) by a TRAF2-dependent mechanism (Yeh et al., 1997). JNK activation is not re- quired for TNF-stimulated apoptosis (Liu et al., 1996; Natoli et al., 1997; Tournier et al., 2000). However, correl- ative studies indicate that JNK signaling may contribute to either apoptotic signaling (De Smaele et al., 2001; Guo et al., 1998; Tang et al., 2001) or antiapoptotic sig- naling (Lee et al., 1997; Roulston et al., 1998) in cells exposed to TNF. The purpose of this study was to identify the role of JNK in apoptosis caused by TNF. Our approach was to compare the response of wild-type and Jnk1 / Jnk2 / (Jnk / ) fibroblasts to TNF. This analysis demonstrated that JNK mediates a survival response in cells treated with TNF. The role of JNK is mediated by the transcrip- tion factor JunD, which can collaborate with NF- B to increase the expression of prosurvival genes, including ciap-2. In the absence of activated NF- B, the JNK pathway mediates an apoptotic response. Together, these data provide a mechanism that can account for the

2 Molecular Cell 1480 Figure 1. Comparison of the TNF-Stimulated Apoptotic Response of Wild-Type and Jnk / Fibroblasts (A) Wild-type and Jnk / fibroblasts cultured in 10% FBS were treated with 10 ng/ml TNF. Extracts prepared at different times following treatment were examined by immunoblot (IB) analysis for JNK. The activity of JNK was measured by an in vitro kinase assay (KA) using c-jun as the substrate. The phosphorylation was detected by autoradiography and was quantitated by PhosphorImager analysis. The number below the autoradiograph of the kinase assays represents the relative kinase activity (control untreated cells 1). The data presented are representative of three independent experiments. (B) Wild-type and Jnk / fibroblasts were incubated in medium supplemented with 10% FBS and 2 M emetine. The cells were treated without or with 10 ng/ml TNF (6 hr). Apoptosis was examined by measurement of DNA fragmentation (mean OD at 405 nm SD; n 3). Control experiments demonstrated that apoptosis was not observed in the absence of TNF or when protein synthesis was partially inhibited using emetine, cycloheximide, or actinomycin D. (C) The time course of cell survival was examined by staining cultures with crystal violet (OD at 590 nm). Wild-type and Jnk / fibroblasts were incubated in medium supplemented with 10% FBS and 1 g/ml cycloheximide. The cells were treated without and with 10 ng/ml TNF. The survival of fibroblasts was examined by crystal violet staining (mean OD at 590 nm SD; n 3). (D) Wild-type and Jnk / fibroblasts were incubated in medium supplemented with 10% FBS and 2 M emetine. The cells were treated without or with 10 ng/ml TNF (6 hr). Cell survival was examined using the MTT assay (mean OD at 570 nm SD; n 3). dual ability of JNK to cause either survival or apoptosis in apoptosis caused by TNF (Figure 1C). Indeed, treatment different cellular contexts. with TNF for 6 hr induced apoptosis of the Jnk / cells, but caused little apoptosis of wild-type cells, (Figure 1D) Results and caused markedly increased apopotic DNA fragmentation in JNK-deficient cells compared with wild-type JNK Signals Survival in Response to TNF cells (Figure 1B). Together, these data indicate that JNK It is known that treatment of cells with TNF causes in- deficiency increases the rate of TNF-stimulated apoptosis. creased JNK activity. To test whether the JNK signaling Thus, JNK provides a survival signal in TNF-stimucreased pathway contributes to TNF responses, we compared lated cells that opposes the effects of TNF in causing the effects of TNF on wild-type and Jnk1 / Jnk2 / apoptosis. (Jnk / ) fibroblasts that lack a functional JNK signaling To examine the relative roles of JNK1 and JNK2 in pathway (Tournier et al., 2000). JNK was detected by the survival response caused by TNF, we compared the immunoblot analysis as a mixture of 46 and 55 kda TNF-stimulated apoptotic response of wild-type, Jnk1 /, isoforms in wild-type cells, but no JNK was observed and Jnk2 / fibroblasts. No differences in TNF-stimu- in Jnk / cells (Figure 1A). As expected, TNF caused lated apoptosis were detected (data not shown). This JNK activation in wild-type fibroblasts, but no JNK activity observation contrasts with the markedly increased apostimulated was detected in JNK-deficient cells. In contrast, TNF- ptosis detected in Jnk / fibroblasts (Figure 1). These p38 MAP kinase activity was detected in both data indicate that JNK1 and JNK2 have largely redun- wild-type and JNK-deficient cells (data not shown). dant roles in the survival signaling response to TNF. JNK-deficient fibroblasts exhibit a profound defect Altered expression of TNF receptors could contribute in TNF-stimulated JNK activity (Figure 1A). Thus, JNK- to the effects of JNK deficiency on TNF responses. We deficient fibroblasts provide a model that can be used therefore examined TNF receptor expression in wild- to critically test whether the JNK signaling pathway type and Jnk / fibroblasts. Ribonuclease protection contributes to TNF-regulated apoptosis. Apoptosis was assays demonstrated that the amount of TNF-R1 mrna examined by measurement of DNA fragmentation. expression in wild-type cells was similar to that detected Treatment of wild-type cells with TNF caused DNA fragmentation. in Jnk / cells (Figure 2A). Similarly, no difference betation Interestingly, TNF-stimulated DNA fragmen- tween wild-type and Jnk / cells was detected when was markedly increased in the Jnk / cells (Figure the amount of cell surface TNF-R1 was examined by 1B). Time course studies indicated that JNK deficiency flow cytometry (Figure 2B). Furthermore, no difference caused an increase in the rate, but not the extent, of in the expression of TNF-R2 was detected between the

3 Mechanism of Survival Signaling by JNK 1481 Figure 2. Comparison of TNF-R1 Expression by Wild-Type and Jnk / Fibroblasts (A) Measurement of TNF-R1 mrna. Fibroblasts were treated (4 hr at 37 C) with 1 g/ml antibody to Fas (Jo2) or 10 ng/ml TNF. Total RNA was examined in a ribonuclease protection assay with probes for TNF-R1 and GAPDH. The mrna were detected by autoradiography (left panel) and were quantitated by PhosphorImager analysis. The relative expression of TNF-R1 compared to GAPDH is presented (right panel). (B) Measurement of the cell surface expression of TNF-R1. Wildtype and Jnk / fibroblasts were examined by flow cytometry using antibodies to TNF-R1. The specific staining of TNF-R1 is presented. Nonspecific staining observed in control experiments is indicated (blue). wild-type and Jnk / cells (data not shown). These data indicate that TNF receptor expression was not altered by JNK deficiency. Figure 3. Complementation Analysis Demonstrates that JNK Re- stores TNF-Stimulated Apoptosis in Jnk / Fibroblasts Expression of JNK Complements the TNF-Stimulated Apoptosis Defect in Jnk / Cells The increased TNF-stimulated apoptosis of Jnk / cells is intriguing. To test whether the altered apoptosis was caused by JNK deficiency, we performed complementation analysis by expression of JNK1 in the Jnk / cells using a retroviral vector. Control experiments were performed using Jnk / cells transduced with the empty retroviral vector. Treatment of wild-type cells with TNF caused a marked increase in JNK activity in wild-type cells, and a lower level of JNK activity was observed in the complemented Jnk / cells (Figure 3A). No JNK activity was detected in Jnk / cells or in Jnk / cells transduced with the empty vector. Markedly increased TNF-stimulated apoptosis of Jnk / cells compared with wild-type cells was detected (Figure 3B). However, transduction of the Jnk / cells with the JNK1 retroviral vector, but not with the empty vector, strongly sup- pressed TNF-stimulated DNA fragmentation (Figure 3B). These data established that the altered TNF-stimulated apoptosis of Jnk / cells was caused by JNK deficiency. In addition, these data demonstrated that JNK acts as an inhibitor of TNF-stimulated apopotosis. (A) Jnk / fibroblasts were transduced with a bicistronic retroviral vector that expressed HA-JNK1 plus ebfp or with a vector that expressed only ebfp. Wild-type fibroblasts, Jnk1 / fibroblasts, and the transduced Jnk1 / fibroblasts were treated with 10 ng/ml TNF (15 min). Cell extracts were examined by immunoblot (IB) analysis for JNK, JunD, and -tubulin. The activity of JNK was measured using an in vitro kinase assay (KA) using c-jun as the substrate. The numbers below the autoradiograph of the phosphorylated c-jun correspond to the relative kinase activity. (B) Complementation with HA-JNK1 restores TNF-stimulated apo- ptosis in Jnk / fibroblasts. Wild-type fibroblasts, Jnk1 / fibroblasts, and Jnk1 / fibroblasts expressing HA-JNK1 plus ebfp (or only ebfp) were incubated in medium supplemented with 10% FBS and 2 M emetine. The cells were treated without and with 10 ng/ml TNF (6 hr). Apoptosis was examined by measurement of DNA fragmentation (mean OD 405 nm SD; n 3). (Beg and Baltimore, 1996; Liu et al., 1996; Van Antwerp et al., 1996; Wang et al., 1996). Decreased function of the NF- B pathway in Jnk / cells could account for the observed increase in TNF-stimulated apoptosis (Figure JNK Is Not Required for TNF-Stimulated 1). We therefore examined the interaction between the NF- B Activation NF- B and JNK pathways. Previous studies have indicated crosstalk between the NF- B and JNK signaling It is established that the NF- B pathway provides an important survival signal in the response of cells to TNF pathways (De Smaele et al., 2001; Reuther et al., 2002;

4 Molecular Cell 1482 Figure 4. JNK Is Not Required for TNF-Stimulated NF- B Transcription Activity (A) Wild-type and Jnk / fibroblasts were incubated with TNF (10 ng/ml) for different times. The amounts of I B- and -tubulin were examined by immunoblot analysis. (B) TNF causes nuclear localization of NF- B in wild-type and Jnk / fibroblasts. The cells were treated without or with 10 ng/ml TNF (1 hr). The nuclear accumulation of p65 NF- B (red) was examined by immunofluorescence analysis (left panels). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, blue) and are shown as merged images (right panels). (C) The NF- B transcription activity in wildtype and Jnk / fibroblasts was examined in transfection assays using a firefly luciferase reporter plasmid containing three NF- B sites cloned upstream of a minimal promoter. The fibroblasts were treated without or with 10 ng/ml TNF (6 hr). Transfection efficiency was monitored using a cotransfected Renilla luciferase expression vector. The normalized NF- B reporter gene expression is presented (mean SD; n 3). Tang et al., 2001). Activation of NF- B causes the ex- not influence the activation of NF- B caused by TNF. pression of one or more genes that downregulate JNK To confirm this conclusion, we examined NF- B activity activity. Consequently, inhibition of NF- B in cells using a luciferase reporter gene in a transient transfection treated with TNF leads to sustained JNK activation. assay. These data demonstrated that TNF caused These data demonstrate that the NF- B pathway regu- increased NF- B transcription activity in both wild-type lates JNK activity. However, it was unclear whether JNK and Jnk / fibroblasts (Figure 4C). Since NF- B isa influenced NF- B activation. We therefore compared potent apoptotic inhibitor, these data indicate that decreased the effects of TNF on the activation of NF- B in wildtype NF- B activation does not account for the inthe and Jnk / cells. Immunoblot analysis demon- creased TNF-stimulated apoptosis of Jnk / cells (Figure strated that the kinetics and extent of I B degradation 1B). caused by TNF were not affected by JNK deficiency (Figure 4A). Immunofluorescence analysis indicated that Role of AKT in JNK-Mediated Survival Signaling p65/rela accumulated in the nucleus of both wild-type JNK deficiency potently increased TNF-stimulated apo- and Jnk / cells following treatment with TNF (Figure ptosis (Figure 1). This increased apoptosis was not mediated 4B). Similarly, electrophoretic mobility shift assays using by changes in cell surface TNF-R1 expression nuclear extracts prepared from wild-type and Jnk / (Figure 2) or by reduced NF- B activation (Figure 4). cells demonstrated that JNK deficiency caused no How might JNK influence cell survival? One possibility change in TNF-stimulated NF- B DNA binding activity is that JNK influences the AKT pathway, which has been (data not shown). These data indicated that JNK does established in previous studies to have a critical role in

5 Mechanism of Survival Signaling by JNK 1483 cell survival (Brunet et al., 2001). We therefore examined cells (Bossy-Wetzel et al., 1997; Ham et al., 1995; Xia et the activation of AKT in wild-type and Jnk / cells. Immunoblot al., 1995), that JunB can antagonize the effects of c-jun analysis demonstrated that the amount of AKT (Deng and Karin, 1993), and that JunD can mediate sur- expressed in wild-type and Jnk / cells was similar (see vival signaling (Weitzman et al., 2000). These considera- Supplemental Figure S1 at tions suggested that increased expression of c-jun or cgi/content/full/11/6/1479/dc1). AKT activation was decreased expression of JunB or JunD may contribute monitored in immunoblots using an antibody that binds to the apoptotic phenotype of JNK-deficient cells. Ribonuclease AKT phosphorylated on the activating site Ser-473. Control protection assays demonstrated that JNK defi- studies of wild-type and Jnk / cells demonstrated ciency did not increase c-jun expression or decrease that serum starvation caused the loss of phospho-ser- JunB expression (data not shown). However, JNK defi- 473 and that acute treatment with insulin or with 10% ciency did cause a marked decrease in JunD mrna serum caused a large increase in AKT phosphorylation expression (Figure 5A). Complementation assays dem- on Ser-473 (data not shown). To examine the role of onstrated that the expression of JNK1 in Jnk / cells AKT under the conditions used for the apoptosis assays, restored the expression of JunD mrna (Figure 5A). we examined AKT phosphorylation in cells grown in These observations were confirmed by immunoblot 10% serum. Treatment of wild-type cells with 1 g/ml analysis using an antibody that binds JunD. JNK deficycloheximide (which causes partial inhibition of protein ciency caused a marked decrease in JunD protein expression, synthesis) caused increased phosphorylation of AKT on and this defect was partially rescued by exsynthesis) Ser-473. Ligation of cell surface Fas using an agonist pression of JNK1 in Jnk / cells (Figure 3A). Together, antibody caused no change in AKT phosphorylation. these data suggest that JNK was required for the normal In contrast, treatment with TNF caused increased AKT expression of JunD in fibroblasts. phosphorylation on Ser-473. Comparative studies using We performed complementation analysis by express- Jnk / cells indicated that basal AKT activation in Jnk / ing JunD in the Jnk / cells (Figure 5B). Immunoblot cells grown in 10% serum was slightly greater than in analysis demonstrated that JNK was detected in wild- wild-type cells. Interestingly, the effects of cyclohexi- type cells, but not in Jnk / cells. Comparison of wildtype mide and TNF to increase AKT activation observed in and Jnk / cells demonstrated that JNK deficiency wild-type cells were not detected in Jnk / cells (see caused reduced JunD expression (Figure 5B). Transduction Supplemental Figure S1 at of the Jnk / cells with a JunD retroviral vector cgi/content/full/11/6/1479/dc1). caused increased JunD expression. To test the role of The reduced TNF-stimulated AKT activation in Jnk / JunD in TNF-stimulated apoptosis, we examined DNA cells may contribute to the effect of JNK deficiency to fragmentation (Figure 5C). As expected, TNF caused cause increased apoptosis. To test whether this defect markedly increased apoptosis of Jnk / cells compared in AKT activation was solely responsible for the in- to wild-type cells. This increased apoptosis was suppressed creased TNF-stimulated apoptosis of Jnk / cells, we in Jnk / cells transduced with the JunD retrovicreased examined the effect of inhibition of PI-3 kinase activity ral vector, but not in Jnk / cells transduced with a using the drug wortmannin (see Supplemental Figure S1 control vector (Figure 5C). Together, these data indicate at that the survival signaling role of JNK can be replaced DC1). Control studies demonstrated that wortmannin by JunD expression. Since JunD is expressed at low abolished AKT phosphorylation on Ser-473 in response levels in Jnk / cells, the role of JNK in survival signaling to serum, insulin, or TNF. If altered regulation of AKT may be to increase JunD expression. was the major mechanism that accounted for the effect of JNK deficiency to increase TNF-stimulated apoptosis, JunD Cooperates with NF- B to Signal Cell Survival we predicted that inhibition of the AKT pathway (using It is most likely that JunD functions by regulating the wortmannin) would eliminate the effects of JNK defi- expression key molecules that act to inhibit TNF-stimuciency on TNF-stimulated apoptosis. This prediction lated apoptosis. Candidate target genes include those was tested by examination of apoptosis using a TUNEL induced by other antiapoptotic signal transduction pathassay. We observed that TNF-stimulated apoptosis in ways, including NF- B (Baud and Karin, 2001). We the presence of wortmannin was markedly increased in tested this hypothesis by examining gene expression Jnk / cells compared with wild-type cells. Together, in wild-type and Jnk / fibroblasts. No defects in the these data indicate that, while altered AKT regulation expression of TRADD, TRAF2, RIP1, FLIP, ciap-1, or may contribute to the effects of JNK deficiency on TNF- caspase (2, 3, 6, 7, 8, and 9) mrna were detected by stimulated apoptosis, the major effects of JNK defi- microarray analysis and ribonuclease protection assays ciency are mediated by an AKT-independent mech- (data not shown). However, ciap-2 was identified as a anism. gene that was selectively induced by TNF only in wildtype cells (Figure 6A). ciap-2 is a caspase inhibitor that Role AP-1 Proteins in JNK-Mediated also interacts with TNF receptor-bound TRAF2 and in- Survival Signaling hibits TNF-stimulated apoptosis (Rothe et al., 1995; Uren It is established that the AP-1 group of transcription et al., 1996). factors represent targets of the JNK signal transduction TNF causes rapid NF- B-dependent ciap-2 expression pathway (Davis, 2000). Substrates of JNK include c-jun, (Chu et al., 1997; Wang et al., 1998). However, JunB, and JunD. These Jun family proteins have distinct ciap-2 mrna expression is also JNK dependent (Figure functions (Jochum et al., 2001). Previous studies have 6A). To confirm this observation, we performed immu- indicated that c-jun can have proapoptotic effects on noblot analysis. JNK deficiency prevented TNF-stimu-

6 Molecular Cell 1484 Figure 5. Complementation with JunD Restores TNF-Stimulated Apoptosis in Jnk / Fibroblasts (A) Complementation with HA-JNK1 restores JunD mrna in Jnk / fibroblasts. Total RNA was isolated from wild-type, Jnk1 / Jnk2 /, and HA-JNK1 complemented Jnk1 / fibroblasts growing in medium supplemented with 10% FBS. The amount of JunD and GAPDH mrna was examined by ribonuclease protection assays. Protected transcripts were resolved by electrophoresis and quantitated by PhosphorImager analysis. JunD mrna expression normalized to GAPDH is presented. (B) Wild-type fibroblasts, Jnk / fibroblasts, and Jnk / fibroblasts transduced with a retroviral vector that expresses JunD in the sense (S) or antisense (AS) orientations were cultured in medium supplemented with 10% FBS. Extracts prepared from the cells were examined by immunoblot (IB) analysis using antibodies to JunD, JNK, and -tubulin. (C) Complementation with JunD restores TNF-stimulated apoptosis in Jnk / fibroblasts. Wild-type fibroblasts, Jnk1 / fibroblasts, and Jnk1 / fibroblasts expressing JunD (A or AS) were treated with 10 ng/ml TNF and 2 M emetine (6 hr). Apoptosis was examined by measurement of DNA fragmentation (mean OD 405 nm SD; n 3). lated expression of ciap-2 (Figure 6B). Interestingly, moter following the exposure of cells to TNF (Hong et TNF-stimulated ciap-2 expression in wild-type cells was al., 2000). To test whether JunD is recruited to the ciap-2 not inhibited during incubation with 1 g/ml cyclohexi- promoter in TNF-treated cells, we performed chromatin mide (Figure 6C), a condition that enables TNF to cause immunoprecipitation (ChIP) assays. Studies of wild-type apoptosis. Furthermore, complementation assays dem- cells demonstrated that c-jun was bound to the ciap-2 onstrated that the expression of JunD in Jnk / cells promoter under basal conditions (Figure 7A). Treatment restored TNF-stimulated expression of ciap-2 (Figure with TNF caused a large increase in JunD binding to the 6D). These data indicate that the loss of ciap-2 gene promoter and a corresponding decrease in the binding expression may contribute to the increased TNF-stimu- of c-jun. These data demonstrate that TNF causes the lated apoptosis observed in JNK-deficient cells. selective recruitment of JunD to the ciap-2 promoter The ciap-2 promoter contains two critical NF- B bind- in wild-type cells. In contrast, studies of Jnk / cells ing sites and also an AP-1 binding site (Hong et al., demonstrated no detectable interaction of c-jun or 2000). To examine the relative roles of NF- B and AP-1 in JunD with the ciap-2 promoter (Figure 7A). Control studciap-2 gene expression, we examined ciap-2 promoter ies demonstrated that TNF caused the recruitment of activity in cotransfection assays using a luciferase re- p65 NF- B to the ciap-2 promoter in both wild-type porter gene (Figure 6E). Treatment with TNF caused a and Jnk / cells (data not shown). Together, these data marked increase in ciap-2 promoter activity. Expression indicate that the cooperation of NF- B with JunD is of dominant-negative I B strongly suppressed, and required for the normal expression of ciap-2 in response dominant-negative Jun partially suppressed, TNF-stim- to treatment with TNF. ulated ciap-2 promoter activity (Figure 6E). These data demonstrated that both NF- B and AP-1 contribute to TNF-stimulated ciap-2 promoter activity. To confirm the Discussion role of JunD in ciap-2 promoter activity, we performed complementation assays. The basal ciap-2 promoter TNF is a multifunctional cytokine that regulates cell pro- activity was similar in wild-type and Jnk / cells (Figure liferation, inflammatory responses, and cell death (Baud 6F). Treatment with TNF caused increased ciap-2 proplex and Karin, 2001). Biological responses to TNF are commoter activity in wild-type cells, but not in Jnk / cells. and reflect the balance of opposing influences of Expression of JunD restored TNF-stimulated ciap-2 the signal transduction pathways that are activated by promoter activity in Jnk / cells (Figure 6F). Together, the TNF receptor. Thus, TNF can cause apoptosis by these data demonstrate that the JNK/JunD pathway colantiapoptotic activating caspase 8. However, TNF also activates the laborates with NF- B to increase the expression of the NF- B pathway. The role of JNK in the antiapoptotic gene ciap-2 in cells exposed to TNF. response to TNF was unclear. Previous studies have demonstrated that JNK activation was not required for TNF Causes Recruitment of JunD TNF-stimulated apoptosis (Liu et al., 1996; Natoli et al., to the ciap-2 Promoter 1997; Tournier et al., 2000), but correlative studies indi- It is established that the ciap-2 promoter contains AP-1 cated that JNK signaling may contribute to apoptotic and NF- B sites and that NF- B is recruited to the pro- signaling (De Smaele et al., 2001; Guo et al., 1998; Tang

7 Mechanism of Survival Signaling by JNK 1485 Figure 6. The JNK/JunD Pathway Is Required for Expression of the Survival Gene c-iap-2 (A) Wild-type and Jnk / cells were treated without and with 10 ng/ml TNF (24 hr). The expression of ciap-2 and GAPDH mrna was examined by hybridization analysis. The normalized data are presented as the mean SD of three independent observations. (B) Wild-type and Jnk / cells were treated without and with 10 ng/ml TNF for the indicated times. Extracts were prepared, and the amount of ciap-1, ciap-2, XIAP, TRAF2, and -tubulin in the cell lysates was measured by immunoblot analysis. (C) The TNF-stimulated expression of ciap-2 by wild-type cells in the presence of 1 g/ml cycloheximide was examined. (D) The TNF-stimulated expression of ciap-2 by Jnk / cells was investigated by immunoblot analysis. The effect of ectopic expression of JunD was examined (Figure 5). (E) Cotransfection assays were performed using a ciap-2 promoter firefly luciferase reporter plasmid in 293T cells to examine the effect of dominant-negative dn-i B and dn-jun. The cells were treated without and with 10 ng/ml TNF (5 hr). Transfection efficiency was examined by cotransfection with a Renilla luciferase reporter plamid, and the data are presented as the normalized ciap-2 promoter activity (mean SD; n 3). (F) ciap-2 promoter activity was examined in wild-type and Jnk / cells treated without and with 10 ng/ml TNF (5 hr). The effect of coexpression of the luciferase reporter vector together with dn-jun or JunD was examined. The data are presented as the normalized ciap-2 promoter activity (mean SD; n 3). JNK Has a Dual Function in Apoptosis Signaling The survival signaling role of JNK in the response to TNF (Figure 1) contrasts with the effects of JNK to mediate apoptosis in response to the exposure of cells to envi- ronmental stress (Davis, 2000). How can one signal transduction pathway mediate two very different re- sponses? There are two general mechanisms that could et al., 2001) or antiapoptotic signaling (Lee et al., 1997; Roulston et al., 1998). In this study, we have directly examined the role of JNK in TNF-stimulated apoptosis by comparing the response of wild-type cells with cells that lack expression of all JNK isoforms. We confirm that JNK is not essential for TNF-stimulated apoptosis. However, we show that the absence of JNK strongly potentiates the apoptotic actions of TNF. Complementation analysis demonstrated that this altered apoptosis in Jnk / cells was caused by JNK deficiency. These data indicate that JNK contributes to the survival response in cells exposed to TNF. This conclusion is consistent with the results of previous studies of Mkk4 gene disruption which causes markedly reduced JNK activity and causes embryonic death mediated by increased TNF-stimulated apoptosis (Ganiatsas et al., 1998; Nishina et al., 1999; Yang et al., 1997). The mechanism of JNK activation caused by TNF involves the adaptor protein TRAF2. It is therefore interesting that TNF does not cause JNK activation in cells derived from Traf2 / mice and that the apoptotic effects of TNF are greatly increased in Traf2 / cells (Yeh et al., 1997). Similar observations have been obtained in studies of cells that express dominant-negative TRAF2 (Lee et al., 1997). Although TRAF2 is implicated in activation of the antiapoptotic NF- B pathway, it appears that Traf2 / cells do not have marked defects in NF- B activation because of redundant functions of TRAF5 (Tada et al., 2001). Thus, it appears that it is the loss of JNK activation rather than changes in NF- B activity that accounts for the increased TNF-stimulated apoptosis of Traf2 / cells.

8 Molecular Cell 1486 account for these different roles of JNK in apoptosis signaling. These mechanisms are not mutually exclusive. One mechanism is represented by the time course of JNK activation. Previous studies of MAP kinases indicate that the time course of activation can determine the cellular response (Traverse et al., 1994). This may also apply to the response of cells to JNK activation. Sustained JNK activation is required for apoptotic signaling (Chen et al., 1996) and is sufficient for apoptosis (Lei et al., 2002). In contrast, TNF causes transient JNK activation (Sluss et al., 1994). These considerations indicate that transient JNK activation (or elevated basal JNK activity) may be important for mediating a survival response in TNF-treated cells and that chronic JNK activation may contribute to apoptotic responses. A second mechanism that may account for the different roles of JNK in apoptosis signaling is that the biological consequence of JNK function may depend upon the activation state of other signal transduction pathways. For example, increased AKT activation can suppress the apoptotic effects of activated JNK (Lei et al., 2002). A plausible hypothesis is that the JNK signaling pathway may cooperate with other signaling pathways to mediate cell survival (e.g., NF- B and AKT). For example, target genes that are induced by the antiapoptotic NF- B pathway may contain JNK-responsive elements in their promoters (e.g., AP-1 sites). The ciap-2 gene represents an example of this class of gene (Figure 7). JNK increases the expression of such genes in cells with activated NF- B and thus increases cell survival (Figure 7). In contrast, in the absence of a survival pathway that can cooperate with JNK, sustained JNK activation may lead to apoptosis (Figure 7). JunD Is a Mediator of Survival Signaling by JNK The observation that Jnk / fibroblasts express low levels of JunD is likely to be biologically significant because this finding may explain at least two phenotypes of JNKdeficient cells. Previous studies have established that JunD / fibroblasts undergo premature senescence in culture that is associated with increased expression of the Mdm2 inhibitor ARF and increased expression of p53 (Weitzman et al., 2000). Interestingly, Jnk / primary fibroblasts also undergo premature senescence that is associated with increased expression of ARF and p53 (Tournier et al., 2000). This similar phenotype of JunD / Figure 7. Regulation of the ciap-2 Promoter by JNK and Jnk / fibroblasts is intriguing. The loss of JunD (A) ChIP assays were performed using wild-type and Jnk / cells expression in JNK-deficient cells may therefore contribtreated without and with 10 ng/ml TNF (24 hr). Specificity controls ute to the properties of Jnk / cells. Decreased NH 2 - for the ciap-2 promoter were performed by immunoprecipitation terminal phosphorylation of JunD in Jnk / cells may without an antibody (upper panel). The interaction of c-jun and also contribute to the observed phenotype. JunD with the ciap-2 promoter was examined (lower panel). The A second phenotype that is shared by JunD / and amplified ciap-2 promoter fragment ( 247/ 1) containing the composite AP-1 and NF- B sites was quantitated after gel electrophoresis by ethidium bromide staining using an AlphaImager 2200v5.5 Jnk / fibroblasts is altered apoptosis. The TNF-stimu- and AlphaEase software (Alpha Innotech Co.), was normalized to the total input, and is presented as relative binding to the promoter. Control studies were performed by amplifying a fragment of the NF- B). One example is represented by ciap-2, a potent antiapo- Gapdh gene. ptotic gene that is strongly induced by TNF. The expression of ciap2 (B) High levels of sustained JNK activation initiate a Bax/Bak-depen- is regulated by the NF- B and AP-1 pathways. In JNK-deficient cells, dent apoptotic pathway that causes the release of cytochrome c loss of JunD expression interferes with the expression of ciap-2. It and activation of the Apaf-1/caspase 9 apoptosome (Lei et al., 2002; is likely that ciap-2 is a member of a larger group of antiapoptotic Tournier et al., 2000). JNK may also contribute to signaling cell genes that are regulated by JNK coordinately with survival signaling survival by cooperating with other survival pathways (e.g., AKT and pathways.

9 Mechanism of Survival Signaling by JNK 1487 lated apoptosis of JunD / cells is markedly potentiated tion mechanisms are employed by many signaling pathways (Weitzman et al., 2000). Similarly, Jnk / cells express to achieve signaling specificity. low levels of JunD and also exhibit increased TNF-stimulated apoptosis (Figure 1). Complementation studies Experimental Procedures demonstrated that the increased apoptosis of Jnk / Descriptions of plasmids and procedures used for immunofluorescells was suppressed by expression of JNK or JunD cence, biochemical analysis, flow cytometry, RNA analysis, reporter (Figures 3 and 5). Thus, the function of JNK to inhibit gene assays, and ChIP assays are provided in the Supplemental apoptosis can be replaced by JunD. Survival signaling Data at by JNK may therefore be mediated by JunD. This function of JunD is consistent with the established role of Tissue Culture this transcription factor as an inhibitor of TNF-stimulated Mouse embryonic fibroblasts (strain 129svJ) were prepared from apoptosis (Weitzman et al., 2000). day 13.5 embryos (Tournier et al., 2000) and were cultured in Dulbec- co s modified Eagle s medium supplemented with 10% fetal calf The mechanism by which JNK increases JunD expresserum (FBS, Invitrogen) and 100 M -mercaptoethanol (Sigma). sion is unclear. Previous studies have demonstrated Recombinant retroviruses were prepared by transient transfection important roles for Oct-1, SP-1, and AP-1 sites in the of plasmids in 293T cells (Pear et al., 1993). Fibroblasts were trans- JunD promoter (de Groot et al., 1991). Transient trans- duced by incubation with the retrovirus ( colony forming fection assays using the JunD promoter fused to a lucif- units/ml) and 8 g/ml polybrene. Transduced cells expressing the erase reporter gene did not indicate a role for Oct-1 or GFP or BFP marker were isolated by flow cytometry. The cells were SP-1 sites. However, the AP-1 site may contribute to treated with TNF (R&D Systems) in medium with 10% FBS. the decreased expression of JunD. This possibility is Measurement of Cell Survival and Apoptosis consistent with the finding that Jnk / cells exhibit se- Cell survival was examined by crystal violet staining (Tournier et verely reduced AP-1 transcription activity (Ventura et al., 2000) and using the methylthiazolyldiphenyl-tetrazolium (MTT) al., 2003). bromide assay following manufacturer s recommendations (Sigma). A key question concerns the identity of the antiapothe TUNEL assays and DNA fragmentation assays were performed using ptotic genes that are regulated by JunD. Previous stud- In Situ Cell Death Detection Kit (TMR red) and the Cell Death Detection Elisa plus kit, respectively, following the manufacturer s reies have identified functional cooperation between p65 commedations (Roche). NF- B and JunD in TNF-stimulated cells (Rahmani et al., 2001). JunD may therefore influence the expression of antiapoptotic genes that are induced by the NF- B Acknowledgments pathway (Chu et al., 1997; Stehlik et al., 1998; Wang et We thank M.J. Birrer and M. Roussel for providing essential plasmids, al., 1998, 1999; Wu et al., 1998). In this study, we identify T. Barrett for constructing the retroviral expression vectors, ciap-2 as a gene that is regulated by the coordinated N.J. Kennedy for discussions, and Kathy Gemme for expert adminis- trative assistance. R.A.F. and R.J.D. are investigators of the Howard actions of JNK/JunD and NF- B. Hughes Medical Institute. This study was supported, in part, by a TNF causes a rapid NF- B-dependent increase in grant from the National Cancer Institute. ciap-2 expression (Chu et al., 1997; Wang et al., 1998). ciap-2 is an inhibitor of caspases that also interacts Received: January 24, 2003 with TNF receptor-bound TRAF2 and causes inhibition Revised: April 8, 2003 of TNF-stimulated apoptosis (Rothe et al., 1995; Uren Accepted: April 9, 2003 et al., 1996). Sequence analysis of the ciap-2 promoter Published: June 19, 2003 indicates the presence of both AP-1 and NF- B binding sites (Hong et al., 2000). Interestingly, TNF-stimulated References ciap-2 expression was not observed in Jnk / cells, Baud, V., and Karin, M. (2001). Signal transduction by tumor necrosis and complementation analysis demonstrated that this factor and its relatives. Trends Cell Biol. 11, defect was restored by expression of JunD (Figure 6). Beg, A.A., and Baltimore, D. (1996). 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