Volume 11, Issue 6, Pages 1479-1489 (June 2003) JunD Mediates Survival Signaling by the JNK Signal Transduction Pathway  Jennifer A. Lamb, Juan-Jose Ventura, Patricia Hess, Richard A. Flavell, Roger J. Davis  Molecular Cell  Volume 11, Issue 6, Pages 1479-1489 (June 2003) DOI: 10.1016/S1097-2765(03)00203-X

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 μM 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 μg/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 μM 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). Molecular Cell 2003 11, 1479-1489DOI: (10.1016/S1097-2765(03)00203-X)

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 μg/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. Wild-type 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). Molecular Cell 2003 11, 1479-1489DOI: (10.1016/S1097-2765(03)00203-X)

Figure 3 Complementation Analysis Demonstrates that JNK Restores TNF-Stimulated Apoptosis in Jnk−/− Fibroblasts (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 α-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 apoptosis 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 μM 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). Molecular Cell 2003 11, 1479-1489DOI: (10.1016/S1097-2765(03)00203-X)

Figure 4 JNK Is Not Required for TNF-Stimulated NF-κB Transcription Activity (A) Wild-type and Jnk−/− fibroblasts were incubated with TNF (10 ng/ml) for different times. The amounts of IκB-α and α-tubulin were examined by immunoblot analysis. (B) TNF causes nuclear localization of NF-κB 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-κB (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-κB transcription activity in wild-type and Jnk−/− fibroblasts was examined in transfection assays using a firefly luciferase reporter plasmid containing three NF-κB 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-κB reporter gene expression is presented (mean ±SD; n = 3). Molecular Cell 2003 11, 1479-1489DOI: (10.1016/S1097-2765(03)00203-X)

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 α-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 μM emetine (6 hr). Apoptosis was examined by measurement of DNA fragmentation (mean OD 405 nm ±SD; n = 3). Molecular Cell 2003 11, 1479-1489DOI: (10.1016/S1097-2765(03)00203-X)

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 α-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 μg/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-IκB 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). Molecular Cell 2003 11, 1479-1489DOI: (10.1016/S1097-2765(03)00203-X)

Figure 7 Regulation of the cIAP-2 Promoter by JNK (A) ChIP assays were performed using wild-type and Jnk−/− cells treated without and with 10 ng/ml TNF (24 hr). Specificity controls for the cIAP-2 promoter were performed by immunoprecipitation without an antibody (upper panel). The interaction of c-Jun and JunD with the cIAP-2 promoter was examined (lower panel). The amplified cIAP-2 promoter fragment (−247/+1) containing the composite AP-1 and NF-κB sites was quantitated after gel electrophoresis by ethidium bromide staining using an AlphaImager 2200v5.5 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 Gapdh gene. (B) High levels of sustained JNK activation initiate a Bax/Bak-dependent apoptotic pathway that causes the release of cytochrome c and activation of the Apaf-1/caspase 9 apoptosome (Lei et al., 2002; Tournier et al., 2000). JNK may also contribute to signaling cell survival by cooperating with other survival pathways (e.g., AKT and NF-κB). One example is represented by cIAP-2, a potent antiapoptotic gene that is strongly induced by TNF. The expression of cIAP2 is regulated by the NF-κB and AP-1 pathways. In JNK-deficient cells, loss of JunD expression interferes with the expression of cIAP-2. It is likely that cIAP-2 is a member of a larger group of antiapoptotic genes that are regulated by JNK coordinately with survival signaling pathways. Molecular Cell 2003 11, 1479-1489DOI: (10.1016/S1097-2765(03)00203-X)