dp5/HRK Is a c-Jun Target Gene and Required for Apoptosis Induced by Potassium Deprivation in Cerebellar Granule Neurons

In cerebellar granule neurons, a BH3-only Bcl-2 family member, death protein 5/harakiri, is up-regulated in a JNK-dependent manner during apoptosis induced by potassium deprivation. However, it is not clear whether c-Jun is directly involved in the induction of dp5. Here, we showed that the up-regulation of dp5, but not fas ligand and bim, after potassium deprivation was suppressed by the expression of a dominant negative form of c-Jun. Deletion analysis of the 5′-flanking sequence of the dp5 gene revealed that a major responsive element responsible for the induction by potassium deprivation is an ATF binding site located at -116 to -109 relative to the transcriptional start site. Mutation of this site completely abolished promoter activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site and increased in potassium-deprived cerebellar granule neurons. Chromatin immunoprecipitation demonstrated that c-Jun was able to bind to this site in vivo. Finally, we demonstrated that knockdown of Dp5 by small interfering RNA rescued neurons from potassium deprivation-induced apoptosis. Taken together, these results suggest that dp5 is a target gene of c-Jun and plays a critical role in potassium deprivation-induced apoptosis in cerebellar granule neurons. In cerebellar granule neurons, a BH3-only Bcl-2 family member, death protein 5/harakiri, is up-regulated in a JNK-dependent manner during apoptosis induced by potassium deprivation. However, it is not clear whether c-Jun is directly involved in the induction of dp5. Here, we showed that the up-regulation of dp5, but not fas ligand and bim, after potassium deprivation was suppressed by the expression of a dominant negative form of c-Jun. Deletion analysis of the 5′-flanking sequence of the dp5 gene revealed that a major responsive element responsible for the induction by potassium deprivation is an ATF binding site located at -116 to -109 relative to the transcriptional start site. Mutation of this site completely abolished promoter activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site and increased in potassium-deprived cerebellar granule neurons. Chromatin immunoprecipitation demonstrated that c-Jun was able to bind to this site in vivo. Finally, we demonstrated that knockdown of Dp5 by small interfering RNA rescued neurons from potassium deprivation-induced apoptosis. Taken together, these results suggest that dp5 is a target gene of c-Jun and plays a critical role in potassium deprivation-induced apoptosis in cerebellar granule neurons. The Bcl-2 family proteins can be divided into three major subgroups (1Huang D.C. Strasser A. Cell. 2000; 103: 839-842Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar). Antiapoptotic proteins, such as Bcl-2, Bcl-XL, and Mcl-1, typically share four conserved motifs termed Bcl-2 homology (BH) 3The abbreviations used are: BH, Bcl-2 homology; Dp5/HRK, death protein 5/haraki; Bim, Bcl2-interacting mediator of cell death; FasL, Fas ligand; CGN, cerebellar granule neuron; JNK, c-Jun NH2-terminal protein kinase; ChIP, chromatin immunoprecipitation; DIV, days in vitro; MOI, multiplicity of infection; NGF, nerve growth factor; Q-PCR, quantitative PCR; PI, propidium iodide; EGFP, enhanced green fluorescent protein. domains and inhibit mitochondrial cytochrome c release and apoptosis. Multidomain proapoptotic proteins, the second subgroup, such as Bax, Bak, and Bok, typically have three BH domains but promote cytochrome c release and apoptosis. The third, and the most structurally diverse subgroup, is the BH3-only proteins, including Dp5/HRK (death protein 5/harakiri), Bim (Bcl2-interacting mediator of cell death), Bid, Bad, Puma, and Noxa, which share the BH3 domain. The BH3-only proteins are critical initiators of apoptosis. Upon challenge, BH3-only proteins translocate to mitochondria and promote the chromec release by neutralizing the antiapoptotic action of Bcl-2 family members. BH3-only proteins are stringently regulated at the transcriptional and post-translational levels during apoptosis, such as Dp5, Bim, and Puma, depending on the cell type and apoptotic stimulus (2Imaizumi K. Tsuda M. Imai Y. Wanaka A. Takagi T. Tohyama M. J. Biol. Chem. 1997; 272: 18842-18848Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 3Inohara N. Ding L. Chen S. Nunez G. EMBO J. 1997; 16: 1686-1694Crossref PubMed Scopus (332) Google Scholar, 4Putcha G.V. Moulder K.L. Golden J.P. Bouillet P. Adams J.A. Strasser A. Johnson E.M. Neuron. 2001; 29: 615-628Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 5Yin K.J. Lee J.M. Chen S.D. Xu J. Hsu C.Y. J. Neurosci. 2002; 22: 9764-9770Crossref PubMed Google Scholar, 6Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Among the BH3-only proteins, Dp5 is of particular interest to studies of apoptosis in the nervous system. In rodents, the expression of Dp5 is largely restricted to and is developmentally regulated in the nervous system (2Imaizumi K. Tsuda M. Imai Y. Wanaka A. Takagi T. Tohyama M. J. Biol. Chem. 1997; 272: 18842-18848Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Kanazawa K. Imaizumi K. Mori T. Honma Y. Tojo M. Tanno Y. Yokoya S. Niwa S. Tohyama M. Takagi T. Wanaka A. Mol. Brain Res. 1998; 54: 316-320Crossref PubMed Scopus (17) Google Scholar). Dp5 is the first found BH3-only protein to be induced by NGF deprivation in sympathetic neurons (2Imaizumi K. Tsuda M. Imai Y. Wanaka A. Takagi T. Tohyama M. J. Biol. Chem. 1997; 272: 18842-18848Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). dp5 is highly homologous to the human gene harakiri (HRK) cloned by a two-hybrid screen with Bcl-2 and Bcl-XL (3Inohara N. Ding L. Chen S. Nunez G. EMBO J. 1997; 16: 1686-1694Crossref PubMed Scopus (332) Google Scholar). As well as being induced in NGF-deprived sympathetic neurons, the induction of dp5 is also observed in cerebellar granule neurons (CGNs) deprived of potassium, cortical neurons exposed to toxic concentrations of amyloid-β protein, retinal ganglion cells of axotomized rat retinas, and axotomized postnatal mouse motoneurons. Overexpression of Dp5 in sympathetic neurons or CGNs induces apoptosis in a Bax-dependent manner, and this effect can be attenuated by co-expression of antiapoptotic Bcl-2. Deletion of dp5 delays sympathetic neuron apoptosis triggered by NGF withdrawal and rescues motoneurons from axotomy-induced apoptosis. These studies suggest that Dp5 plays a critical role in neuronal apoptosis. Studies from several laboratories have demonstrated that JNK is involved in dp5 up-regulation during neuronal injury or apoptosis. In sympathetic neurons deprived of NGF (8Harris C.A. Johnson Jr., E.M. J. Biol. Chem. 2001; 276: 37754-37760Abstract Full Text Full Text PDF PubMed Google Scholar), CGNs deprived of potassium (8Harris C.A. Johnson Jr., E.M. J. Biol. Chem. 2001; 276: 37754-37760Abstract Full Text Full Text PDF PubMed Google Scholar), cortical neurons exposed to amyloid-β protein (9Bozyczko-Coyne D. O'Kane T.M. Wu Z.L. Dobrzanski P. Murthy S. Vaught J.L. Scott R.W. J. Neurochem. 2001; 77: 849-863Crossref PubMed Scopus (159) Google Scholar), and spinal cord injury triggered by trauma (10Yin K.J. Kim G.M. Lee J.M. He Y.Y. Xu J. Hsu C.Y. Neurobiol. Dis. 2005; 20: 881-889Crossref PubMed Scopus (40) Google Scholar), dp5 is induced in a manner that is clearly JNK-dependent. Although JNK has been shown to be involved in dp5 up-regulation, the mechanism of how JNK regulates dp5 expression is not clarified. In the present study, we used recombinant adenoviruses that express a dominant negative form of c-Jun (FLAG-Δ169) to identify the potential targets of c-Jun in neuronal apoptosis. We showed that c-Jun mediates the induction of dp5, but not that of fas ligand (fasL) and bim during potassium deprivation-induced apoptosis in CGNs. In addition, we identified an ATF site located at -116 to -109 of the dp5 promoter that was required for its activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site, and the association of c-Jun/ATF2 complex was significantly increased in potassium-deprived CGNs. Chromatin immunoprecipitation (ChIP) demonstrated that c-Jun was able to bind to this site in vivo. Finally, knockdown of Dp5 protects CGNs from apoptosis. Taken together, our results suggest that dp5 is a c-Jun target gene and required for apoptosis induced by potassium deprivation in CGNs. Neuronal Culture and Potassium Deprivation—Rat CGNs were prepared from 7-8-day-old Sprague-Dawley rat pups (15-19 g) as described previously (11Li M. Wang X. Meintzer M.K. Laessig T. Birnbaum M.J. Heidenreich K.A. Mol. Cell. Biol. 2000; 20: 9356-9363Crossref PubMed Scopus (335) Google Scholar, 12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar). Briefly, neurons were dissociated from freshly dissected cerebella by mechanical disruption in the presence of trypsin and DNase and then seeded at a density of 1.5 × 106 cells/ml in basal modified Eagle's medium containing 10% fetal bovine serum and 25 mm KCl (25 K+S). Cytosine arabinoside (10 μm) was added 24 h after seeding to limit the growth of nonneuronal cells. For potassium deprivation, experiments were performed as described previously (11Li M. Wang X. Meintzer M.K. Laessig T. Birnbaum M.J. Heidenreich K.A. Mol. Cell. Biol. 2000; 20: 9356-9363Crossref PubMed Scopus (335) Google Scholar, 12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar). Briefly, cells cultured for 7 days (DIV7) in medium containing 25 K+S were switched into serum-free medium containing 25 or 5 mm KCl (25 K or 5 K) in the presence or absence of the inhibitors SP600125 (Calbiochem) or CEP11004 (a kind gift from Cephalon Inc.). Cells that did not receive inhibitors received Me2SO as a control. The final concentration of Me2SO was less than 0.1%. Western Blotting—Western blotting analysis was performed as described in detail previously (11Li M. Wang X. Meintzer M.K. Laessig T. Birnbaum M.J. Heidenreich K.A. Mol. Cell. Biol. 2000; 20: 9356-9363Crossref PubMed Scopus (335) Google Scholar, 12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar). Briefly, protein lysates prepared from neurons were separated by a 10% polyacrylamide gel, transferred to polyvinylidene difluoride membrane and subjected to immunoblotting with polyclonal antibodies against phospho-JNK (Thr183/Tyr185), JNK, phospho-c-Jun (Ser73) (Cell Signaling Technology; diluted 1:1000), and c-Jun (610327; BD Biosciences Pharmingen; diluted 1:1000) and monoclonal antibodies to FLAG or tubulin (Sigma; both diluted 1:10,000) at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized using the ECL chemiluminescence system (Amersham Biosciences). Q-PCR—Total RNA was extracted and isolated from CGNs using TRIzol reagent (Invitrogen) as described previously (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar). First strand cDNA was synthesized from 1 μg of mRNA by using SuperscriptIII reverse transcriptase (Invitrogen) and oligo(dT) as primers. Q-PCR was performed in triplicate on an ABI Prism 7000 sequence detection system using ABI Sybr Green PCR mixture as described by the manufacturer. PCR cycling conditions were as follows: initial denaturation at 95 °C for 5-10 min followed by 40 cycles of 95 °C for 30 s, 1 min of annealing (annealing temperature adapted for the specific primer set used), and 1 min of extension at 72 °C. Fluorescence data were collected during the annealing stage of amplification. Specificity of the amplification was verified by melt curve analysis. Cycle threshold (Ct) values were calculated using identical threshold values for all experiments. β-Actin was used as control and for normalization. Relative RNA expression was calculated using the formula ratio = 2(Ctref -Cttarget). Data shown represent the mean and S.E. of three separate experiments. The following primer pairs were used: c-jun forward (5′-TGGGCACATCACCACTACAC-3′) and reverse (5′-AGTTGCTGAGGTTGGCGTA-3′); dp5 forward (5′-AGACCCAGCCCGGACCGAGCAA-3′) and reverse (5′-ATAGCACTGAGGTGGCTATC-3′); bim forward (5′-CTACCAGATCCCCACTTTTC-3′) and reverse (5′-GCCCTCCTCGTGTAAGTCTC-3′); fasL forward (5′-CCACCTCCATCACCACTACC-3′) and reverse (5′-GCTGGGGTTGGCTATTTG-3′); β-actin forward (5′-CAACTGGGACGATATGGAGAAG-3′) and reverse (5′-TCTCCTTCTGCATCCTGTCAG-3′). Reporter Construction and Luciferase Assays—A fragment spanning from -1568 to +81 relative to the transcription start site of rat dp5 genomic sequence was produced by PCR with the forward primer 5′-CCATCTAGCTAGCTAGCATCTATTA-3′ and the common reverse primer 5′-AGCGTCGCCGCAACC-3′. This fragment was fused to the promoterless firefly luciferase gene of pGL3-Basic vector (Promega) to generate a dp5 (-1568/+81)-luc. A series of reporter constructs with the same 3′-end but different 5′-ends were also constructed by PCR using dp5 (-1568/+81)-luc as the template and the common reverse primer as mentioned above. Forward primers are as follows: 5′-ATCTTTTCTTATAACAGCCGAA-3′ for dp5 (-356/+81)-luc; 5′-GAGCCTCCCGCAGCC-3′ for dp5 (-270/+81)-luc; 5′-CGGGCCGGATGATGTAA-3′ for dp5 (-125/+81)-luc; and 5′-CCCCCTCCACCATGTGACACTT-3′ for dp5 (-85/+81)-luc. The mutated ATF site (TTACATCA to GGACATCG) (13Ueno Y. Kume N. Miyamoto S. Morimoto M. Kataoka H. Ochi H. Nishi E. Moriwaki H. Minami M. Hashimoto N. Kita T. FEBS Lett. 1999; 457: 241-245Crossref PubMed Scopus (16) Google Scholar) was introduced into dp5 (-1568/+81)-luc by overlap extension PCR. First, two DNA fragments with a 19-bp overlap (GAGGGGGGGACATCGTCCG) containing the mutated ATF site were synthesized by PCR using dp5 (-1568/+81)-luc as template with forward primer 5′-GATAGGTACCCCATCTAGCTAGCTAGCATCTATTA-3′ and reverse primer 5′-GAGGGGGGGACATCGTCCG-3′ and with forward primer 5′-CGGACGATGTCCCCCCCTC-3′ and reverse primer 5′-CTCGAGATCTAGCGTCGCCGCAACC-3′. The full length of the -1568 to +81 fragment of dp5 promoter containing the mutated ATF site was obtained by mixing the two DNA fragments produced from the first-step PCR as template in the second PCR with the outermost primers. The second PCR was carried out at 94 °C for 1 min and then 25 cycles of denaturing at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 3 min. All PCR products were subcloned into the NheI and HindIII sites of pGL3-Basic. The sequence encoding FLAG-Δ169 was amplified from the Ad-FLAG-Δ169 by PCR and subcloned into pcDNA3 between XhoI and HindIII following the ATG and 8-amino acid FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). Specific primers are as follows: forward, 5′-ATTACTCGAGATGGACTACAAGGACGA-3′; reverse, 5′-TATAAAGCTTTTAAAACGTTTGCAACTGCT-3′. All of the constructs were confirmed by DNA sequencing. CGNs were transfected using the calcium phosphate co-precipitation method as described previously (11Li M. Wang X. Meintzer M.K. Laessig T. Birnbaum M.J. Heidenreich K.A. Mol. Cell. Biol. 2000; 20: 9356-9363Crossref PubMed Scopus (335) Google Scholar, 12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar). Plasmids transfected contained 2 μg of various reporter plasmids, 9 μg of expression plasmids or pcDNA3-based vectors, and 100 ng of Renilla luciferase reporter plasmid, pCMV-RL (Promega). Neurons were kept in conditioned medium after transfection for 12 h and then switched to the serum-free medium containing 25 or 5 mm KCl for 12 h. The levels of firefly luciferase activity were normalized to Renilla luciferase activity, as we reported previously (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar). Adenovirus Infection—The recombinant adenoviruses, Ad-GFP and Ad-FLAG-Δ169 (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar, 14Whitfield J. Neame S.J. Paquet L. Bernard O. Ham J. Neuron. 2001; 29: 629-643Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar), were purified and used as described previously (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar). DIV5 neurons were infected with Ad-GFP or Ad-FLAG-Δ169 at a multiplicity of infection (MOI) of 100 for 36 h. To test the efficiency of adenovirus-mediated gene transfer, CGNs were infected at DIV5 with 100 MOI of Ad-FLAG-Δ169, the cells were fixed 36 h after infection and stained with FLAG antibody and with the DNA dye bisbenzimide (Hoechst 33258) (5 μg/ml) to visualize nuclear morphology. Fluorescent images were captured by using a fluorescence microscope equipped with a CCD camera. Gel Mobility Shift Assay—Nuclear extracts were prepared as described in detail previously (15Li M. Linseman D.A. Allen M.P. Meintzer M.K. Wang X. Laessig T. Wierman M.E. Heidenreich K.A. J. Neurosci. 2001; 21: 6544-6552Crossref PubMed Google Scholar). Synthetic oligonucleotide probes spanning the ATF binding site (forward, 5′-AGCTCGGATGATGTAACCCCGATC-3′; reverse, 5′-GATCGGGGTTACATCATCCGAGCT-3′), as well as those bearing mutated ATF site (forward, 5′-AGCTCGGACGATGTCCCCCCGATC-3′; reverse, 5′-GATCGGGGGGACATCGTCCGAGCT-3′) were annealed and labeled with γ-32P (PerkinElmer Life Sciences) by using T4 polynucleotide kinase. 32P-Labeled probes were incubated with 5 μg of nuclear proteins in a 20-μl DNA binding reaction buffer. For supershift, 1 μg of c-Jun antibody (catalog number sc-1694x; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 1 μg ATF2 antibody (catalog number sc-187x; Santa Cruz Biotechnology) was preincubated with nuclear extracts at 4 °C for 1 h. DNA protein complexes were resolved by 4% polyacrylamide gel and exposed to photography. ChIP Assay—ChIP was performed using the ChIP assay kit (Upstate Cell Signaling Solutions) according to the manufacturer's instructions. Approximately 4.5 × 107 cerebellar granule neurons were used in each treatment. 4 h after they were switched to the serum-free medium containing 25 or 5 mm KCl, neurons were cross-linked by the addition of 1% formaldehyde for 10 min at room temperature and were terminated with glycine (final concentration of 0.125 m). Neurons were harvested and incubated in 600 μl of SDS lysis buffer containing protease inhibitors for 10 min on ice. Chromatins were sonicated to yield fragments of about 0.5 kb in length. After sonication, the lysate was centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was diluted in ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-HCl, pH 8.1, 167 mm NaCl, and protease inhibitors). After it was precleared with protein G-agarose, 5% of the supernatant was saved as input DNA. 2 μg of rabbit c-Jun antibody (catalog number sc-1694x; Santa Cruz Biotechnology) or rabbit normal IgG (Sigma) was added to the supernatant and incubated overnight at 4 °C with rotation. After wash, immune complexes were eluted with elution buffer (1% SDS, 0.1 m NaHCO3, and 200 mm NaCl). A part of the captured immunocomplex was subjected to Western blotting with another mouse c-Jun antibody (610327; BD Biosciences Pharmingen) to detect whether captured chromatins contained c-Jun. Cross-linking was reversed by heating at 65 °C overnight. RNA was degraded with RNase A for 30 min, and protein was degraded with proteinase K for 2 h. DNA was purified using the EZChIP polypropylene spin column and subjected to PCR amplification using the primers spanning the ATF site on the dp5 promoter (forward, 5′-AGGGTTAAAAGTTACCTCTCGGC-3′; reverse, 5′-ACCCCAAGTTTCGCTCTGC-3′; c-jun promoter spanning the TRE-jun2 site (forward, 5′-CTAGACAGCCAAACCAAGAC-3′; reverse, 5′-GCTCACGGGATGAGGTAAT-3′). RNA Interference—The BS/U6 vector was kindly provided by Dr. Cress W. Douglas (University of South Florida College of Medicine) (16Ma Y. Cress W.D. Haura E.B. Mol. Cancer Ther. 2003; 2: 73-81PubMed Google Scholar). To ensure the specificity, multiplicity controls were employed (17Nat. Cell Biol. 2003; 5: 489-490Crossref PubMed Scopus (163) Google Scholar). Two Dp5 small hairpin RNAs (shRNAs), shdp5a and shdp5b, targeting the 19-nucleotide AGAGAAACGGGATGTCATT (938-956) or GACGGAGCGTGATTTCTAA (1386-1404) in the 3′-untranslated region of dp5 mRNA (NCBI accession number NM_057130) were designed (18Kalinec G.M. Fernandez-Zapico M.E. Urrutia R. Esteban-Cruciani N. Chen S. Kalinec F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16019-16024Crossref PubMed Scopus (63) Google Scholar). The targeted regions showed no significant homology with any other genes by BLAST searches. The shRNA expression cassettes containing sense loop (TTCAAGAGA)-antisense termination signal T6 were inserted downstream of the U6 promoter. For ectopic Dp5 expression, the intact coding sequence of dp5 with its 3′-untranslated region containing the shRNA targeting sites was amplified from the genome of Sprague-Dawley rats and inserted into pCMV3×FLAG (Sigma). All constructs were confirmed by sequencing. To knock down the expression of FLAG-dp5, 0.5 μg of FLAG-dp5 and 5 μg of shdp5a or shdp5b were co-transfected into 293A cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. 48 h after transfection, cell lysates were harvested and processed for Western blotting with FLAG antibody to detect the expression of FLAG-dp5 protein. DIV5 CGNs were transfected with BS/U6 vector, shdp5a, or shdp5b together with pCMV-EGFP by the calcium phosphate co-precipitation method (11Li M. Wang X. Meintzer M.K. Laessig T. Birnbaum M.J. Heidenreich K.A. Mol. Cell. Biol. 2000; 20: 9356-9363Crossref PubMed Scopus (335) Google Scholar). EGFP was used to mark the transfected cells. 48 h after transfection, neurons were switched to medium containing 25 or 5 mm KCl for 12 h. Neurons were stained with Hoechst 33258 to visualize nuclear morphology and propidium iodide (PI) to detect membrane damage. Apoptosis was quantified by scoring the percentage of EGFP-positive neuron population with pyknotic nuclei or with PI-positive cells. Unbiased counting cells (>600 for each group) were scored blindly without knowledge of their previous treatment. JNK Mediates the Up-regulation of dp5 during Apoptosis Induced by Potassium Deprivation in CGNs—To determine whether potassium deprivation evokes the activation of JNK/c-Jun in CGNs, DIV7 neurons maintained in 25 K+S were switched to 25 K or 5 K medium for various durations of time (0.5, 1, 2, and 4 h), and then processed for Western blotting with antibodies against phospho-JNK and phospho-c-Jun. Consistent with previous studies (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar, 19Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar), potassium deprivation led to an increase in the level of phospho-JNK starting at 0.5 h without detectable changes in total JNK level (Fig. 1A). As observed in Fig. 1A and consistent with previous results (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar), potassium deprivation also resulted in a significant increase in levels of c-Jun phosphorylation starting at 0.5 h and lasting for up to 4 h, which paralleled the increase in level of JNK phosphorylation. c-Jun is a autoregulated immediate early gene, and our results clearly showed that the c-Jun protein level is significantly increased at 0.5 h. These results suggested that JNK and c-Jun are activated in the early period of apoptosis induced by potassium deprivation, as we reported previously (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar). To test whether the observed potassium deprivation-evoked increase of JNK/c-Jun activity was accompanied by the induction of dp5, a time course experiment was carried out in which dp5 mRNA levels were measured through Q-PCR analysis at different times (0.5, 1, 2, and 4 h). The dp5 mRNA increased significantly, starting at ∼1 h after the time point (0.5 h) at which JNK/c-Jun activation was detected after potassium deprivation (Fig. 1B). To clarify whether JNK activity is required for dp5 up-regulation, we first used a JNK inhibitor, SP600125 (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar, 20Bennett B.L. Sasaki D.T. Murray B.W. O'Leary E.C. Sakata S.T. Xu W. Leisten J.C. Motiwala A. Pierce S. Satoh Y. Bhagwat S.S. Manning A.M. Anderson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13681-13686Crossref PubMed Scopus (2244) Google Scholar). Treatment of CGNs with SP600125 inhibited the induction of dp5 mRNA by ∼62% (Fig. 1C). As a further test of specificity, SP600125 was replaced by CEP11004, a mixed lineage kinase inhibitor that inhibits the JNK pathway but not JNK itself. Treatment with CEP11004 led to an over 65% reduction of dp5 mRNA (Fig. 1C). These results suggest that JNK is involved in dp5 up-regulation. The incomplete inhibition of dp5 induction by SP600125 or CEP11004 was not due to their inefficiency to inhibit JNK, because the induction of c-jun mRNA, a result of JNK activation, was completely inhibited in both inhibitor-treated cultures (Fig. 1C). Consistent with a previous study (8Harris C.A. Johnson Jr., E.M. J. Biol. Chem. 2001; 276: 37754-37760Abstract Full Text Full Text PDF PubMed Google Scholar), these results indicate that JNK partially mediates the up-regulation of dp5 by potassium deprivation in CGNs. Dominant Negative c-Jun, FLAG-Δ169, Attenuates the Up-regulation of dp5—The facts that c-Jun, a downstream transcription factor of JNK, was activated after potassium deprivation (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar, 19Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar, 21Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Crossref PubMed Google Scholar) preceding the up-regulation of dp5 and that inhibition of the JNK/c-Jun pathway attenuated dp5 induction (Fig. 1B) raise the possibility that the activation of c-Jun is required for the up-regulation of dp5. To identify the c-Jun-dependent events in the neuronal apoptosis and investigate whether c-Jun is involved in dp5 up-regulation, we used the recombinant adenoviruses expressing FLAG-Δ169 (Ad-FLAG-Δ169) (12Shi L. Gong S. Yuan Z. Ma C. Liu Y. Wang C. Li W. Pi R. Huang S. Chen R. Han Y. Mao Z. Li M. Neurosci. Lett. 2005; 375: 7-12Crossref PubMed Scopus (22) Google Scholar, 14Whitfield J. Neame S.J. Paquet L. Bernard O. Ham J. Neuron. 2001; 29: 629-643Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 22Ham J. Babij C. Whitfield J. Pfarr C.M. Lallemand D. Yaniv M. Rubin L.L. Neuron. 1995; 14: 927-939Abstract Full Text PDF PubMed Scopus (757) Google Scholar), a c-Jun dominant negative mutant, to inhibit c-Jun activity. Without the transactivation domain, it prevents endogenous c-Jun complex from activating target genes by exhausting functional c-Jun and occupying c-Jun binding sites. To verify that FLAG-Δ169 encoded by the adenoviruses was expressed in infected CGNs, the cells were fixed 36 h after infection and stained with FLAG antibody and with the DNA dye Hoechst 33258 to visualize nuclear morphology. Fig. 2A showed representative immunofluorescence pictures of neurons infected with Ad-FLAG-Δ169 at an MOI of 100, and ∼70-80% of the CGNs were found to express FLAG-Δ169. c-Jun is a nuclear protein, and FLAG-Δ169 was also localized exclusively in the nucleus. We also detected the expression of Ad-Δ169 by Western blotting with FLAG antibody (Fig. 2A). In order to test whether c-Jun mediates the induction of dp5 mRNA by potassium deprivation observed here, the CGNs were infected at DIV5 with 100 MOI of Ad-FLAG-Δ169 or Ad-GFP and then were maintained in 25 K+S for 36 h. The infected neurons were switched to medium containing 25 or 5 mm KCl for 4 h. RNA was isolated, and Q-PCR experiments were performed using primers specific for c-jun, dp5, bim, fasL, or β-actin. Potassium deprivation led to a significant increase in c-jun, dp5, bim, and fasL mRNA level in cells infected with Ad-GFP. It is well characterized that c-jun is an autoregulated gene, its protein c-Jun binds to the TRE-jun1 and TRE-jun2 sites on its own promoter to promote its expression (23Angel P. Hattori K. Smeal T. Karin M. Cell. 1988; 55: 875-885Abstract Full Text PDF PubMed Scopus (995) Google Scholar). Results showed that Ad-FLAG-Δ169 almost completely in