Activating Transcription Factor 3 (ATF3) Promotes Sublytic C5b-9-Induced

G Model IMBIO-50689; No. of Pages 11 ARTICLE IN PRESS

Immunobiology xxx (2011) xxx–xxx

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Immunobiology

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Activating transcription factor 3 (ATF3) promotes sublytic C5b-9-induced glomerular mesangial cells apoptosis through up-regulation of Gadd45␣ and KLF6 gene expression

Kuanfeng Xu, Ying Zhou, Wen Qiu, Xin Liu, Mei Xia, Lisha Liu, Xiaomei Liu, Dan Zhao, Yingwei Wang ∗

Department of Microbiology and Immunology, Nanjing Medical University, 140 Nanjing Hanzhong Road, Nanjing, PR China article info abstract

Article history: The sublytic C5b-9 complexes can result in glomerular mesangial cells (GMCs) apoptosis, which involved Received 29 November 2010 in the initiation and development of rat Thy-1 nephritis. Activating transcription factor 3 (ATF3) is an Received in revised form 13 January 2011 immediate early gene for cells to cope with a variety of stress signals, and our previous study revealed that Accepted 15 February 2011 ATF3 could promote GMCs apoptosis attacked by sublytic C5b-9. But the mechanism of ATF3 promoting GMCs apoptosis triggered by sublytic C5b-9 attack has not been elucidated. In this study, the data showed Keywords: that the expression of ATF3, growth arrest and DNA damage-45 alpha (Gadd45␣), Krüppel-like factor 6 Sublytic C5b-9 (KLF6) and proliferating cell nuclear antigen (PCNA) in the GMCs in response to sublytic C5b-9 stimulation ATF3 Gadd45␣ for the indicated time was signiﬁcantly increased, and ATF3 expression could lead to GMCs apoptosis ␣ ␣ KLF6 through up-regulation of Gadd45 and KLF6, but not up-regulation of PCNA. Furthermore, Gadd45 was PCNA identiﬁed as a downstream target gene regulated by ATF3 directly, and KLF6 might be regulated by ATF3 in an indirect manner. Crown Copyright © 2011 Published by Elsevier GmbH. All rights reserved.

Introduction ment in the initiation or development of the nephritis (Amore and Coppo 2000; Qiu et al. 2009; Shimizu et al. 2000; Xu et al. 2006), but Mesangioproliferative glomerulonephritis (MsPGN), character- the mechanisms governing GMCs apoptosis during the early stage ized by damage of glomerular mesangial cells (GMCs) and excessive of Thy-1N remain unclear. secretion of extracellular matrix (ECM), is a disease of high inci- Activating transcription factor 3 (ATF3), a member of the dence in humans (Berthoux et al. 2008; Yamamoto and Wilson ATF/cyclic AMP response element-binding (CREB) family, contains 1987). Rats Thy-1 nephritis (Thy-1N) is a well-known model of a basic region/leucine zipper DNA-binding motif and binds to the human MsPGN (Yamamoto and Wilson 1987), and complement is ATF/CRE consensus sequence TGACGTCA (Hai and Hartman 2001). generally regarded as the principal mediator of GMCs lesions in Overwhelming evidence indicates that ATF3 can be induced rapidly the rats with Thy-1N (Bohana-Kashtan et al. 2004; Brandt et al. by a variety of stress stimuli in different cell types (Ameri et al. 2007; 1996). It has been reported that injury to nucleated cells by comple- Jiang et al. 2010; Kawauchi et al. 2002; Kool et al. 2003; Mungrue ment C5b-9 is almost non-lytic (sublytic) because of homologous et al. 2009; Tamura et al. 2005; Turchi et al. 2009; Yin et al. 2008). restriction factors on the cell surface, such as CD59 and MCP. Pre- Although ATF3 has dual effects on cell fate, i.e., apoptosis (Cai et al. vious studies have revealed that sublytic C5b-9 can lead to GMCs 2000; Fan et al. 2002; Kang et al. 2003; Li et al. 2008; Lu et al. 2006; apoptosis/necrosis and secondary proliferation in the progression Turchi et al. 2009) or cell proliferation (Allan et al. 2001; Kawauchi of Thy-1N (Brandt et al. 1996; Cole and Morgan 2003; Gao et al. et al. 2002; Nakagomi et al. 2003; Perez et al. 2001; Tamura et al. 2006; Qiu et al. 2009; Shimizu et al. 2000; Wang et al. 2006). There 2005), our results showed that the expression of ATF3 was signiﬁ- is growing interest in studying GMCs apoptosis and their involve- cantly increased both in renal tissue of rats with Thy-1N in vivo and in cultured primary rat GMCs induced by sublytic C5b-9 in vitro, and ATF3 was involved in promoting rat GMCs apoptosis attacked by sublytic C5b-9 (Jiang et al. 2010). Abbreviations: MsPGN, mesangioproliferative glomerulonephritis; GMCs, glomerular mesangial cells; ECM, extracellular matrix; Thy-1N, Thy-1 nephritis; ATS, Despite sublytic C5b-9 triggering and ATF3 promoting GMCs anti-thymocyte serum; HIS, heat-inactivated serum; ATF3, activating transcription apoptosis, the mechanisms of ATF3 enhancing GMCs apoptosis factor 3; Gadd45␣, growth arrest and DNA damage-45 alpha; KLF6, Krüppel-like have not been fully elucidated. As a transcription factor, ATF3 must factor 6; PCNA, proliferating cell nuclear antigen; Dox, doxcycline; ChIP, chromatin exert its action, at least partly, by regulating downstream tar- immunoprecipitation. ∗ get genes, such as IL-6, C/EBP homologous protein, NOXA, CCL4, Corresponding author. Tel.: +86 25 86862769; fax: +86 25 86508960. E-mail address: [email protected] (Y. Wang). ﬁbronectin 1, and so on (Khuu et al. 2007; Litvak et al. 2009;

Mungrue et al. 2009; Wang et al. 2009; Yin et al. 2008). However, Construction of Gadd45˛ and KLF6 promoter no target genes of ATF3 in sublytic C5b-9-induced GMCs apoptosis have been identified. Therefore, the purpose of this study was to Constructs of Gadd45␣ and KLF6 luciferase promoter in paren- explore the mechanism involved in rat GMCs apoptosis, includ- thesis relative to the transcriptional start site were generated by ing identifying downstream target genes of ATF3 regulation in the polymerase chain reaction of rat genomic DNA using primers listed GMCs apoptosis attacked by sublytic C5b-9. in Table S1. Products were transferred into pGL3-basic luciferase reporter vector in the appropriate direction using the enzymes Hind III/Kpn I, ligated with T4 DNA ligase, and sequence-verified. Materials and methods GMCs culture and sublytic C5b-9 determination Reagents ◦ Rat GMCs were cultured at 37 C, 5% CO2 conditions and main- Polyclonal antibodies against ATF3 (sc-188), Gadd45␣ (sc- tained in MEM supplemented with 10% FBS, and used from five to 6850), KLF6 (sc-20085) and ␤-actin (sc-47778) were purchased seven passages for experiments. The formation of sublytic C5b-9 on from Santa Cruz Biotechnology (USA), monoclonal antibody against GMCs membrane was identified as previously described (Gaoetal. PCNA was from Thermo scientific company (USA). PrimeScriptTM 2006, 2009; Qiu et al. 2009). The selection of ATS and complement RT reagent kit and SYBR Premix Ex TaqTM were from Takara (Japan). concentration used in this study was 4% ATS and 4% NS with less Annexin V/PI apoptosis detection kit was from Biouniquer Technol- than 5% LDH release or cell lysis. To demonstrate that the effects ogy Co., Ltd. (USA). Luciferase assay kits were from Promega (USA). on GMCs following above stimulation were in fact due to sublytic ChIP analysis kits were from Upstate (Charlottesville, VA). Plasmids C5b-9, control GMCs were treated with MEM, 4% ATS, 4% ATS + 4% Tet-off advanced, TRE-tight-ATF3 and doxycycline (Dox) were from HIS and 4% ATS + 4% C6dS (Gao et al. 2006; Qiu et al. 2009; Cybulsky Clontech (USA). et al. 2005; Fosbrink et al. 2005). The rat glomerular mesangial cell line (HBZY-1) was purchased from China Centre for Type Culture Collection (Wuhan, China). Nor- GMCs infection, transfection and identification mal human serum (NS) from healthy adult donors were pooled and used as a source of serum complement, and heat-inactivated An effective AdMaxTM-Tet off advanced system to regulate ATF3 serum (HIS) was prepared by incubating at 56 ◦C for 30 min. Human expression in the GMCs was established, and the cultured GMCs complement C6-deficient serum (C6dS) was purchased from Sigma were infected with this system for the indicated time (4 h, 12 h, (USA). Rabbit polyclonal antibody against Thy-1 antigen of rat thy- 24 h and 48 h) or different doses of Dox to demonstrate the role mocyte, namely the anti-thymocyte serum (ATS), was prepared of ATF3 in the GMCs. To further investigate the role of ATF3 in the according to previously published procedures (Gao et al. 2006; Qiu GMCs induced by sublytic C5b-9, the cultured GMCs were infected et al. 2009; Xu et al. 2006). Ad-MaxTM-GFP, adenovirus reconstruc- with AdMaxTM-Tet-off advanced system for 42 h, then attacked by tion and purification were offered by Vector Gene Technology Co., sublytic C5b-9 for 6 h. Experimental groups were as follows: MEM, Ltd. (China). Generation of SiGadd45␣, SiKLF6 and RNAi-Mate were Ad-vec (Ad-TRE-tight-ATF3), Ad-ATF3 (Ad-Tet off + Ad-TRE-tight- offered by Shanghai GenePharma Co., Ltd. (China). ATF3), Dox (Ad-Tet off + Ad-TRE-tight-ATF3 plus 1 ␮g/mL Dox), sublytic C5b-9, Ad-vec + sublytic C5b-9, Ad-ATF3 + sublytic C5b-9, Dox + sublytic C5b-9. Moreover, ATF3 ShRNA expression plasmids Construction of AdMaxTM-mediated Tet-off and TRE-tight-ATF3 were transiently transfected into the cultured GMCs, and the ShRNA that could effectively silence the ATF3 gene was chosen as our study To obtain AdMaxTM-mediated Tet off and TRE-tight-ATF3, mentioned previously (Jiang et al. 2010). pDC312-Tet-off were reconstructed with plasmids Tet off advanced Besides, three classes of SiRNA to different target sites of and adenoviral shuttle plasmids DC312, and pDC316-TRE-tight- Gadd45␣ or KLF6 were designed (Table S2). And SiGadd45␣ and ATF3 were reconstructed with plasmids TRE-tight-ATF3 and SiKLF6 were transiently transfected into the cultured rat GMCs with adenoviral shuttle plasmids DC316. After sequence-verified, RNAi-Mate according to the manufacturer’s instructions, respec- pDC312-Tet-off and pDC316-TRE-tight-ATF3 were co-transfected tively, subsequently treated with sublytic C5b-9 for 6 h. The optimal with pBHGlox E1, 3Cre into HEK293 cells, respectively, to produce target sequence to silence Gadd45␣ or KLF6 was selected (Fig. S1). recombinant AdMaxTM named Ad-Tet-off and Ad-TRE-tight-ATF3. Then Ad-Tet-off and Ad-TRE-tight-ATF3 were purified by high per- Real-time PCR analysis formance liquid chromatography (HPLC), and tested for replication competent adenoviruses (RCA) contamination by RT-PCR. The quantification of ATF3, Gadd45␣, KLF6 and PCNA expression in the fixed time was performed using real-time PCR through ABI PRISM 7300 sequence detection system. Rat GAPDH was Generation of ShATF3, SiGadd45˛ and SiKLF6 co-amplified as an internal control. The primers were listed in Table S3. The relative level of gene expression was obtained by To ensure the silencing of target genes, different ShRNA calculating ratio of cycle numbers of the initial exponential ampli- sequences were designed to be homologous to ATF3 consensus fication phase as determined by the sequence detection system sequence, and different SiRNA sequences were designed to be for specific target gene and GAPDH using the following formula: homologous to Gadd45␣ and KLF6 consensus sequence. Generation 2(normalized CT in control sample − normalized CT in stimulated sample). of ATF3 ShRNA-expressing vector was described according to our previous study (Jiang et al. 2010). The sequence of Gadd45␣ from Western blot analysis the Rattus norvegicus growth arrest and DNA damage-inducible 45 alpha, complete cds (NM 024127.2) was chosen as SiGadd45␣ tar- The cultured GMCs were homogenized with lysis buffer. Pro- get gene. The sequence of KLF6 from the R. norvegicus Krüppel-like teins from each sample were assayed by SDS–PAGE using a 15% factor 6, complete cds (NM 031642.2) was chosen as SiKLF6 tar- SDS–PAGE gel, then were transferred to PVDF membranes and get gene. The complementary oligonucleotides encoded a hairpin probed with anti-ATF3 (diluted 1:800), anti-Gadd45␣ (diluted structure with a 21-mer stem derived from the target site. 1:600), anti-KLF6 (diluted 1:600) or anti-PCNA (diluted 1:1000)

Fig. 1. Expression of ATF3, Gadd45␣, KLF6 and PCNA in the GMCs attacked by sublytic C5b-9 stimulation. The cultured rat GMCs were treated with sublytic C5b-9 for the indicated time, and analyzed by real-time PCR (A) and Western blot (B) for ATF3, Gadd45␣, KLF6 and PCNA mRNA and protein levels. (A) The results showed that ATF3, Gadd45␣ and KLF6 mRNA levels in sublytic C5b-9-induced GMCs were up-regulated at 40 min (P < 0.05), peaked at 3 h (P < 0.01), then decreased at 6 h (P < 0.05), and PCNA mRNA expression was up-regulated slightly at 3 h and 6 h (P < 0.05). (B) Photomicrographs showing ATF3, Gadd45␣, KLF6 and PCNA protein expression in sublytic C5b-9- induced GMCs for the indicated time. Semi-quantitative analysis displayed that ATF3 protein expression in the GMCs attacked by sublytic C5b-9 emerged at 3 h (P < 0.05), markedly increased at 6 h (P < 0.01), then decreased at 12 h and 24 h. KLF6 and Gadd45␣ protein expression became markedly increased at 3 h and 6 h (P < 0.01), then gradually decreased at 12 h and 24 h, but PCNA protein expression obviously increased at 6 h and 12 h (P < 0.05), and reached a peak at 24 h (P < 0.01). (C) Levels of ATF3, Gadd45␣, KLF6 and PCNA mRNA in sublytic C5b-9 group at 6 h were signiﬁcantly higher than those in MEM, ATS, ATS + HIS and ATS + C6dS groups. (D) Photomicrographs showing ATF3, Gadd45␣, KLF6 and PCNA protein expression at 6 h in the groups mentioned-above. Semi-quantitative analysis displayed that ATF3, Gadd45␣, KLF6 and PCNA protein levels in sublytic C5b-9 group were markedly up-regulated, compared with other groups. ␤-Actin was used as control for mRNA and protein level respectively. All data represented averages and mean ± SD from three independent experiments. Comparisons between different groups were analyzed by ANOVA and post hoc Bonferroni correction (*P < 0.01 vs. MEM, P < 0.01 vs. ATS,P < 0.01 vs. ATS + HIS, #P < 0.01 vs. ATS + C6dS).

Fig. 2. Regulation of Gadd45␣, KLF6 and PCNA protein levels in ATF3-expressing GMCs. (A) Photomicrographs of ATF3, Gadd45␣, KLF6 and PCNA protein expression in the GMCs infected with AdMaxTM-Tet off system for the indicated time by Western blot. Semi-quantitative analysis displayed that ATF3, Gadd45␣ and KLF6 protein levels in the GMCs infected with Ad-ATF3 were markedly increased at 24 h and 48 h (*P < 0.05, **P < 0.01), compared with Ad-vec group for the same treatment, and PCNA protein level displayed no statistic signiﬁcance between two groups. Ad-vec represented for Ad-Tet off or Ad-TRE-tight-ATF3, Ad-ATF3 represented for Ad-Tet off + Ad-TRE-tight-ATF3. (B) ATF3, Gadd45␣, KLF6 and PCNA protein expression in the GMCs infected with AdMaxTM-Tet off system and treated with different doses of Dox for 48 h by Western blot. Semi-quantitative analysis showed that ATF3, Gadd45␣ and KLF6 protein levels were decreased gradually with increasing doses of Dox, compared with AdMaxTM-Tet

Chromatin immunoprecipitation (ChIP) assay Regulation of Gadd45˛, KLF6 and PCNA gene in the GMCs expressed ATF3 ChIP assays were performed as the protocol by Upstate. GMCs 7 TM (2 × 10 cells) were infected with AdMax -Tet off system at m.o.i. To explore the role of ATF3 in the signaling cascade that connects 75 for 48 h, then GMCs were cross-linked with 1% formaldehyde for sublytic C5b-9 attack and expression of Gadd45␣, KLF6 and PCNA, 10 min. After washing twice with PBS, the cells were collected and an AdMaxTM-Tet-off system to regulate ATF3 expression by differ- lysed with 2 ml of SDS lysis buffer containing a protease inhibitor ent doses of Dox was established (Fig. S3A and B). Using this system, mixture, and sonicated to DNA lengths of 200–500 bp. After cen- the expression of Gadd45␣ and KLF6 in ATF3-expressing GMCs was trifugation at 13,000 rpm for 10 min, supernatants were diluted 10 up-regulated, but PCNA expression was not altered (Fig. 2A), and fold in ChIP dilution buffer. Immunoprecipitations were then per- when ATF3 expression was gradually decreased in the GMCs via formed with ATF3 antibody. ChIP DNA was detected using standard increasing dose of Dox, the expression of Gadd45␣ and KLF6 was PCR with the following primer pairs for different regions of the gradually reduced, but PCNA expression was not changed (Fig. 2B). Gadd45␣ and KLF6 promoter, as listed in Table S4. Additionally, ATF3 expression could indeed promote GMCs apoptosis (Fig. S3C). Statistical analysis To further confirm the effect of ATF3 on regulation of Gadd45␣ and KLF6, the cultured GMCs were infected with AdMaxTM-Tet All data were summarized as mean ± SD. One-way ANOVA with off system, and subsequently stimulated by sublytic C5b-9. The simultaneous multiple comparisons between groups by the Bon- results displayed that ATF3 protein level in the GMCs treated ferroni method was used, and P < 0.05 was considered significant. with Ad-ATF3 + sublytic C5b-9 was significantly higher than other All experiments were performed in triplicate. treatments, and Gadd45␣ and KLF6 protein levels also markedly increased in the GMCs treated with Ad-ATF3 + sublytic C5b-9 than Results other treatments (Fig. 2C). Furthermore, the number of apoptotic GMCs in Ad-ATF3 + sublytic C5b-9 group was significantly higher Expression of ATF3, Gadd45˛, KLF6 and PCNA in the GMCs than those in Ad-ATF3, sublytic C5b-9, Ad-vec + sublytic C5b- attacked by sublytic C5b-9 9, Dox + sublytic C5b-9 groups, although these groups displayed increased GMCs apoptosis compared with MEM, Ad-vec or Dox To investigate the expression of pathogenic genes in the GMCs group (Fig. S4A). Changes of Gadd45␣, KLF6 and PCNA protein lev- attacked by sublytic C5b-9, microarray experiments were per- els after sublytic C5b-9 attack were also monitored in the GMCs

off system alone (*P < 0.05, **P < 0.01), but PCNA expression showed no statistical significance in each group. (C) ATF3, Gadd45␣, and KLF6 protein expression in the GMCs treated with AdMaxTM-Tet off system and sublytic C5b-9 complexes by Western blot. ATF3 over-expressing GMCs and groups attacked by sublytic C5b-9 displayed significant up-regulations of the expression of ATF3, Gadd45␣, and KLF6 compared with MEM, Ad-vec or Dox group (*P < 0.05, **P < 0.01), furthermore, in ATF3 over-expressing GMCs (Ad-Tet off + Ad-TRE-tight-ATF3) + sublytic C5b-9 was significantly higher than those in ATF3 over-expressing GMCs or sublytic C5b-9 attack alone (P < 0.01). (D) ATF3, Gadd45␣, KLF6 and PCNA protein expression in the GMCs treated with ShATF3. The protein levels of ATF3, Gadd45␣, and KLF6 in ShATF3 + sublytic C5b-9 group were significantly lower than those in the ShControl + sublytic C5b-9 group (*P < 0.05). ␤-Actin was used as control for protein level. All data represent as mean ± SD from three independent experiments.

Please cite this article in press as: Xu, K., et al., Activating transcription factor 3 (ATF3) promotes sublytic C5b-9-induced glomerular mesangial cells apoptosis through up-regulation of Gadd45␣ and KLF6 gene expression. Immunobiology (2011), doi:10.1016/j.imbio.2011.02.005 G Model IMBIO-50689; No. of Pages 11 ARTICLE IN PRESS 6 K. Xu et al. / Immunobiology xxx (2011) xxx–xxx transfected with ATF3 shRNA. In the GMCs transfected with ShATF3 Role of Gadd45˛ or KLF6 in sublytic C5b-9-induced GMCs and treated with sublytic C5b-9 for 6 h, the ATF3 induction was apoptosis nearly completely ablated. Both Gadd45␣ and KLF6 protein levels were decreased and maintained a lower level in the GMCs To further assess the role of Gadd45␣ in GMCs apoptosis medi- transfected with ShATF3, but not PCNA protein level (Fig. 2D). ated by sublytic C5b-9, GMCs were transfected with SiGadd45␣ Moreover, our results also found that the number of apoptotic for 16 h to knock down the expression of Gadd45␣ gene. After GMCs in ShATF3 + sublytic C5b-9 group was significantly lower the GMCs stimulated by sublytic C5b-9 for 6 h, the protein level than that in ShControl + sublytic C5b-9 group (Fig. S4B). The results of Gadd45␣ was determined by Western blot. The results mani- indicated that both endogenous and exogenous expression of ATF3 fested that the level of Gadd45␣ protein in the GMCs transfected in the GMCs could up-regulate expression of Gadd45␣ and KLF6 with SiGadd45␣ following sublytic C5b-9 stimulation was signif- gene, and ATF3 was involved in promoting GMCs apoptosis induced icantly decreased in comparison with SiControl + sublytic C5b-9 by sublytic C5b-9 attack. (Fig. 5A). And GMCs apoptosis was also significantly decreased in SiGadd45␣ + sublytic C5b-9 group, which indicated that Gadd45␣ gene knockdown in the GMCs could notably suppress GMCs apo- Direct target genes of ATF3 in the GMCs induced by sublytic C5b-9 ptosis induced by sublytic C5b-9 attack (Fig. 5B). Interestingly, the expression of ATF3, KLF6 and PCNA was not altered when Gadd45␣ Although the results mentioned-above showed an altered expression in the GMCs was silenced (Fig. 5A). expression of Gadd45␣ and KLF6 in ATF3-expressing GMCs, they To evaluate the effect of KLF6 on GMCs apoptosis, GMCs were did not indicate whether Gadd45␣ and KLF6 were direct target transfected with SiKLF6 to knock down the expression of KLF6. The genes of ATF3 or not. To address this issue, Gadd45␣ and KLF6 gene results demonstrated that KLF6 expression was not only silenced promoter sequences were analyzed using the MotifScanner. Both successfully in SiKLF6 + sublytic C5b-9 group (Fig. 5 C), but also Gadd45␣ and KLF6 contained putative binding sites for CREB motif: GMCs apoptosis was decreased, indicating that KLF6 gene knock- Gadd45␣ at position −122/−133, and KLF6 at position −198/−209 down in the GMCs could also markedly inhibit GMCs apoptosis relative to the transcriptional start site, respectively. However, for triggered by sublytic C5b-9 (Fig. 5D). However, when KLF6 gene some promoters, the sites are more than 2 kilobases (kb) upstream expression was silenced, the expression of ATF3, Gadd45␣ and from the transcriptional start (+1) site, but the potential binding PCNA gene was not altered (Fig. 5C). site within 250 base pair (bp) from the +1 site was focused in our experiments. In order to elucidate the effect of ATF3 on Gadd45␣ gene Discussion transcription directly, luciferase assay was performed. Compared with control treatments, significant enhancement of luciferase Early studies reported that in the early phase of Thy-1N model, activity was observed in the GMCs transfected with Gadd45␣ GMCs are targeted by anti-Thy-1 antibody resulting in immedi- promoter including predicted CREB site, in which ATF3 expres- ate complement activation and cell injury (Brandt et al. 1996), and sion was induced by sublytic C5b-9, but there was no statistical GMCs apoptosis/necrosis is considered to be a contributor to the significance of luciferase activity in the cells transfected with initiation of Thy-1 nephritis (Amore and Coppo 2000; Qiu et al. Gadd45␣ promoter without the predicted CREB site (Fig. 3A). 2009; Shimizu et al. 2000; Xu et al. 2006; Yamamoto and Wilson These results were consistent with those in ATF3-expressing GMCs 1987). In this study, we confirmed that sublytic C5b-9 complexes infected with AdMaxTM-Tet off system (Fig. 3B), suggesting that could indeed lead to GMCs apoptosis. Although we had implicated ATF3 could activate the Gadd45␣ promoter. Next, to identify that GMCs apoptosis induced by sublytic C5b-9 in rats with Thy- whether ATF3 binds to the predicted CREB site of Gadd45␣ or 1N was dependent on Gadd45␥ up-regulation in part (Qiu et al. not, ChIP analysis was also performed, cross-linked and sheared 2009), the molecular mechanisms responsible for GMCs apoptosis chromatin was immunoprecipitated with antibody against ATF3. of Thy-1N, especially sublytic C5b-9-induced GMCs apoptosis, have The specificity of the band was demonstrated by lack of sig- not been well elucidated. nals when control immunoglobulin (IgG) or 1 ␮g/mL Dox. The It is well known that ATF3 could play a critical role in mediat- precipitated DNA was amplified with primers encompassing the ing cellular apoptosis or proliferation, and the diversity in the final proximal region of Gadd45␣ (Fig. 3C). Over-expression of ATF3 readouts is most likely to be determined by the context of cells was shown in −Dox group (Fig. 3D), which resulted in a ChIP sig- (Chen et al. 2008; James et al. 2006; Mungrue et al. 2009). In our nal delivered from two different primers flanking the CREB-like experiments, we found that ATF3 mRNA and protein levels in the binding site on the Gadd45␣ promoter, and no ChIP signal was GMCs attacked by sublytic C5b-9 were remarkably increased, and detected in +Dox group (Fig. 3E). Taken all together, these results ATF3 expression could indeed promote GMCs apoptosis triggered revealed that Gadd45␣ is a bona fide target gene of ATF3 in rat by sublytic C5b-9 attack. And our results also showed that Gadd45␣ GMCs. and KLF6 were significantly up-regulated and co-expressed with To demonstrate the effect of ATF3 on KLF6 gene transcription, ATF3. In order to explore the mechanism of GMCs apoptosis medi- luciferase assay was also done. However, whether KLF6 promoter ated by sublytic C5b-9 stimulation and to identify downstream including the predicted CREB site or not, no significant difference target genes of up-expressed ATF3 in the GMCs exposed to sub- of luciferase activity was found in all treated GMCs (Fig. 4A), which lytic C5b-9, we analyzed Gadd45␣ and KLF6 promoter sequences was consistent with that in ATF3-expressing GMCs infected with using the MotifScanner, and found that both of them including the AdMaxTM-Tet off system (Fig. 4B). Furthermore, ChIP analysis was predicted ATF/CRE binding site. preformed to identify the binding ability of ATF3 to the predicted As the most well known gene in the Gadd45 family (including CREB site of KLF6 promoter (Fig. 4C). Over-expression of ATF3 was Gadd45␣, Gadd45␤, Gadd45␥), Gadd45␣ is a small acidic protein found in −Dox group, but the results did not show a significant consisting of 165 amino acids and is preferentially localized in the ChIP signal derived from two different primers flanking the CREB- nuclei. Indeed, the majority of the known Gadd45␣ interacting like binding site on KLF6 promoter, neither in −Dox group nor in molecules are nuclear functional proteins, such as p21cip1, xero- +Dox group (Figs. 3D and 4D). These results suggested that ATF3, derma pigmentosum complementation group G and so on (Barreto which regulated KLF6 gene expression, might be dependent upon et al. 2007; Kearsey et al. 1995). Interaction of Gadd45␣ with these an indirect manner. proteins causes cell cycle arrest, repression of DNA replication or

Fig. 3. Activity analysis of Gadd45␣ gene promoter and binding of ATF3 to Gadd45␣ gene promoter in ATF3-expressing GMCs. (A) The cultured rat GMCs were transiently transfected with equal amounts of Gadd45␣ promoter constructs (pGL3-basic-Gadd45␣-1 and pGL3-basic-Gadd45␣-2 included the potential ATF/CRE binding sites, pGL3- basic-Gadd45␣-3 did not include) and PRL-SV40 for the indicated time, then treated with sublytic C5b-9 for 6 h, then the cells were assayed for luciferase activity. Significant enhancement of luciferase activity was observed in the ATF3-expressing GMCs induced by sublytic C5b-9, which transfected with the pGL3-basic-Gadd45␣-1 or pGL3-basic- Gadd45␣-2 (*P < 0.01 vs. MEM, P < 0.01 vs. ATS, P < 0.01 vs. ATS + HIS, #P < 0.01 vs. ATS + C6dS), but not transfected with pGL3-basic-Gadd45␣-3. (B) The cultured rat GMCs were co-transfected with Gadd45␣ promoter constructs and PRL-SV40 for the indicated time, then infected with AdMaxTM-Tet off system. Significant increase of luciferase activity was observed in the −Dox group (Ad-Tet off + Ad-TRE-tight-ATF3), which transfected with pGL3-basic-Gadd45␣-1 or pGL3-basic-Gadd45␣-2 (*P < 0.01 vs. Ad-Tet off + Ad-TRE-tight-ATF3 plus 1 ␮g/mL Dox), but not transfected with the pGL3-basic-Gadd45␣-3. (C) Schematic of Gadd45␣: the transcriptional start (+1), potential ATF/CRE binding sites and the primer target sites were indicated. (D and E) The cultured GMCs were infected with Ad-Tet off + Ad-TRE-tight-ATF3 or Ad-Tet off + Ad-TRE-tight-ATF3 puls 1 ␮g/mL Dox for 48 h. Immunoprecipitated DNA was amplified by PCR for the proximal promoter regions of Gadd45␣ gene. The results displayed that ATF3 protein level in ATF3-expressing GMCs was significantly higher than that in 1 ␮g/mL Dox treated GMCs (*P < 0.01), and there was a significant ChIP signal in the ATF3-expressing GMCs, derived from two different primers flanking the CREB-like binding site on Gadd45␣ promoter. cell apoptosis. Extensive efforts have also been made to understand control transcription of Gadd45␣ gene independently from p53 how the transcription of Gadd45␣ is regulated in cellular responses to suppress mammary tumors (Maekawa et al. 2008). In current to extracellular signals. The transcriptional induction of Gadd45␣ study, either endogenous or exogenous expression of ATF3 could by DNA damage is thought to be dependent on p53 (Zerbini et al. indeed increase Gadd45␣ expression in the GMCs attacked by sub- 2005) and forkhead transcription factor 3a (FoxO3a) (Brunet et al. lytic C5b-9. On the other hand, our results suggested that ATF3 2004), which antagonizes transcriptional repressors of Gadd45␣ over-expression in rat GMCs could obviously elevate Gadd45␣ pro- gene, such as ZBRK (Zheng et al. 2000). Furthermore, one study moter activity, and ATF3 could bind to the predicted CREB site of indicates that ATF2, another member of ATF/CRBE family, could Gadd45␣ promoter at position −122/−133. Besides, the apoptotic

Fig. 4. Activity analysis of KLF6 gene promoter and binding of ATF3 to KLF6 gene promoter in ATF3-expressing GMCs. (A and B) The cultured rat GMCs were transiently transfected with equal amounts of KLF6 promoter constructs (pGL3-basic-KLF6-1 included potential ATF/CRE binding sites, pGL3-basic-KLF6-2 did not include) and PRL- SV40 for the indicated time, and then treated with sublytic C5b-9 complexes or infected with AdMaxTM-Tet off system. The cells were assayed for luciferase activity. No significant enhancement of luciferase activity was observed in neither sublytic C5b-9-induced nor AdMaxTM-Tet off system regulated ATF3-expressing GMCs, which transfected with pGL3-basic-KLF6-1 or pGL3-basic-KLF6-2. Values were mean ± SD and expressed as fold increase relative to similarly transfected controls. (C) Schematic of KLF6: the transcriptional start (+1), potential ATF/CRE binding sites and the primer target sites were indicated. (D) The cultured GMCs were infected with AdMaxTM-Tet off system for 48 h. Cells were harvested for ATF3 protein expression, and ChIP assay was performed using anti-ATF3 antibody or control IgG. Immunoprecipitated DNA was amplified by PCR for the proximal promoter regions of KLF6 gene. The results showed that ATF3 protein level in Ad-Tet off + Ad-TRE-tight-ATF3 group was significantly higher than that in the 1 ␮g/mL Dox treated GMCs (P < 0.01), but ATF3 did not bind directly to KLF6 promoter. All data were from three independent experiments. ratio of GMCs in response to sublytic C5b-9 attack decreased signif- KLF6, another potential downstream target gene of ATF3, is a icantly when Gadd45␣ gene was silenced. These results indicated zinc-finger nuclear protein that belongs to the Krüppel-like family that Gadd45␣ gene might be downstream target gene of ATF3. In of transcription factors. KLF6 contains three C2H2 zinc fingers at the other words, ATF3 could play an important role in promoting GMCs C-terminal domain, recognizes GC or CACCC motifs, and is broadly apoptosis triggered by sublytic C5b-9 complexes directly through expressed in numerous cell types at various developmental stages up-regulation of Gadd45␣ gene. (Slavin et al. 1999). KLF6 activation can result in cell cycle arrest,

Fig. 5. Effects of Gadd45␣ or KLF6 on sublytic C5b-9-induced GMCs apoptosis. (A) Numbers of apoptotic GMCs treated by SiGadd45␣ were analyzed by flow cytometry. The number of apoptotic GMCs in SiGadd45␣ + sublytic C5b-9 group was significantly lower than that in SiControl + sublytic C5b-9 group (*P < 0.05). (B) ATF3, Gadd45␣, KLF6 and PCNA protein expression in the GMCs treated by SiGadd45␣. The protein level of Gadd45␣ in the SiGadd45␣ + sublytic C5b-9 group was significantly lower than that in the SiControl + sublytic C5b-9 group (*P < 0.05), but protein level of ATF3, KLF6 and PCNA showed no statistical significance between two groups. (C) Numbers of apoptotic GMCs treated with SiKLF6 were analyzed by flow cytometry. The number of apoptotic GMCs in SiKLF6 + sublytic C5b-9 group was significantly lower than that in SiControl + sublytic C5b-9 group (*P < 0.05). (D) ATF3, Gadd45␣, KLF6 and PCNA protein expression in the GMCs treated with SiGadd45␣. The protein levels of KLF6 in SiKLF6 + sublytic C5b-9 group were significantly lower than that in SiControl + sublytic C5b-9 group (*P < 0.05), but protein level of ATF3, Gadd45␣ and PCNA showed no statistical significance between two groups.

Please cite this article in press as: Xu, K., et al., Activating transcription factor 3 (ATF3) promotes sublytic C5b-9-induced glomerular mesangial cells apoptosis through up-regulation of Gadd45␣ and KLF6 gene expression. Immunobiology (2011), doi:10.1016/j.imbio.2011.02.005 G Model IMBIO-50689; No. of Pages 11 ARTICLE IN PRESS 10 K. Xu et al. / Immunobiology xxx (2011) xxx–xxx decreased angiogenesis and invasion. Recent study also demon- References strates that the role of transfected exogenous KLF6 in suppressing tumor growth of lung cancer cells is due to cell apoptosis (Ito et al. Allan, A.L., Albanese, C., Pestell, R.G., LaMarre, J., 2001. Activating transcription factor 3 induces DNA synthesis and expression of cyclin D1 in hepatocytes. J. Biol. 2004). As a transcription factor, KLF6 targets include transforming Chem. 276, 27272–27280. growth factor (TGF)-␤1, TGF-␤ receptor, urokinase plasminogen Ameri, K., Hammond, E.M., Culmsee, C., Raida, M., Katschinski, D.M., Wenger, R.H., activator, inducible nitric oxide synthase and so on (Kojima et al. Wagner, E., Davis, R.J., Hai, T., Denko, N., Harris, A.L., 2007. Induction of activating transcription factor 3 by anoxia is independent of p53 and the hypoxic HIF 2000; Warke et al. 2003). However, little information is available signalling pathway. Oncogene 26, 284–289. regarding cellular factors involved in regulating KLF6 gene expres- Amore, A., Coppo, R., 2000. Role of apoptosis in pathogenesis and progression of sion. In our experiment, we revealed that either endogenous or renal diseases. Nephron 86, 99–104. exogenous expression of ATF3 could up-regulate KLF6 gene expres- Barreto, G., Schäfer, A., Marhold, J., Stach, D., Swaminathan, S.K., Handa, V., Döderlein, G., Maltry, N., Wu, W., Lyko, F., Niehrs, C., 2007. Gadd45␣ promotes epigenetic sion in the GMCs exposed to sublytic C5b-9. However, our further gene activation by repair-mediated DNA demethylation. Nature 445, 671–675. results suggested that ATF3 over-expression in rat GMCs could Berthoux, F.C., Mohey, H., Afiani, A., 2008. Natural history of primary IgA nephropa- not increase KLF6 promoter activity, and could not bind to the thy. Semin. Nephrol. 28, 4–9. Bohana-Kashtan, O., Ziporen, L., Donin, N., Kraus, S., Fishelson, Z., 2004. Cell signals predicted CREB site of KLF6 promoter. Moreover, GMCs apoptosis transduced by complement. Mol. Immunol. 41, 583–597. induced by sublytic C5b-9 decreased significantly when KLF6 was Brandt, J., Pippin, J., Schulze, M., Hansch, G.M., Alpers, C.E., Johnson, R.J., Gordon, K., silenced. Taken together, ATF3 promoted sublytic C5b-9-mediated Couser, W.G., 1996. Role of the complement membrane attack complex (C5b-9) in mediating experimental mesangioproliferative glomerulonephritis. Kidney GMCs apoptosis might be due to up-regulation of KLF6 in an indirect Int. 49, 335–343. manner. Here, it was worthy to note that our results were inconsis- Brunet, A., Sweeney, L.B., Sturgill, J.F., Chua, K.F., Greer, P.L., Lin, Y., Tran, H., Ross, S.E., tent with Huang’s data, in which ATF3 is a key target gene of KLF6 Mostoslavsky, R., Cohen, H.Y., Hu, L.S., Cheng, H.L., Jedrychowski, M.P., Gygi, S.P., Sinclair, D.A., Alt, F.W., Greenberg, M.E., 2004. Stress-dependent regulation of in tumor suppression, and ATF3 can mediate KLF6-induced cancer FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011–2015. cells apoptosis under stress conditions (Huang et al. 2008). Based on Cai, Y., Zhang, C., Nawa, T., Aso, T., Tanaka, M., Oshiro, S., Ichijo, H., Kitajima, S., 2000. our data, we thought that the different results might associate with Homocysteine-responsive ATF3 gene expression in human vascular endothelial cells: activation of c-Jun NH2 terminal kinase and promoter response element. initiating distinct signal pathways under different stimulations and Blood 96, 2140–2148. different cell types. Chen, S.C., Liu, Y.C., Shyu, K.G., Wang, D.L., 2008. Acute hypoxia to endothelial cells In addition, PCNA, which plays a central role in cell cycle reg- induces activating transcription factor 3 (ATF3) expression that is mediated via ulation, was also up-regulated in sublytic C5b-9-induced GMCs. nitric oxide. Atherosclerosis 201, 281–288. Cole, D.S., Morgan, B.P., 2003. Beyond lysis: how complement influences cell fate. Previous study has demonstrated that ATF3 directly regulates the Clin. Sci. (Lond.) 104, 455–466. PCNA-associated factor KIAA0101/p15PAF (Turchi et al. 2009). Using Cybulsky, A.V., Takano, T., Papillon, J., Bijian, K., Guillemette, J., 2005. Activation of the MotifScanner, we found that PCNA also contained putative the extracellular signal-regulated kinase by complement C5b-9. Am. J. Physiol. − − Renal Physiol. 289, F593–F603. binding sites for CREB at position 189/ 199 relative to the tran- Fan, F., Jin, S., Amundson, S.A., Tong, T., Fan, W., Zhao, H., Zhu, X., Mazzacurati, L., Li, scriptional start site. But our further experiments showed that ATF3 X., Petrik, K.L., Fornace Jr., A.J., Rajasekaran, B., Zhan, Q., 2002. ATF3 induction expression in rat GMCs did not regulate PCNA expression, and not following DNA damage is regulated by distinct signaling pathways and over- expression of ATF3 protein suppresses cell growth. Oncogene 21, 7488–7496. bind to the predicted CREB site of PCNA promoter. As PCNA reached Fosbrink, M., Niculescu, F., Rus, H., 2005. The role of C5b-9 terminal complement a peak level in the GMCs treated with sublytic C5b-9 later at 24 h, complex in activation of the cell cycle and transcription. Immunol. Res. 31, we considered that PCNA expression might be related to GMCs 37–46. Gao, L., Qiu, W., Wang, Y., Xu, W., Xu, J., Tong, J., 2006. Sublytic complement C5b-9 secondary proliferation. Interestingly, some reports indicate that complexes induce thrombospondin-1 production in rat glomerular mesangial Gadd45␣ appears to interact with PCNA through its flexible loop cells via PI3-k/Akt, association with activation of latent transforming growth (Sánchez et al. 2010; Vairapandi et al. 2000), which suggests a role factor-␤1. Clin. Exp. Immunol. 144, 326–334. ␣ Gao, L., Zhang, Y., Qiu, W., Xu, W., Feng, X., Ren, J., Jiang, X., Wang, H., Zhao, D., Wang, of Gadd45 gene might be involved in regulating PCNA function, Y., 2009. Effects of PI3-k/Akt short hairpin RNA on proliferation, fibronectin but not PCNA expression. production and synthesis of thrombospondin-1 and transforming growth factor- In conclusion, our results indicated that increase of ATF3 beta1 in glomerular mesangial cells induced by sublytic C5b-9 complexes. Cell expression had a promoting role in GMCs apoptosis triggered Prolif. 42, 83–93. Hai, T., Hartman, M.G., 2001. The molecular biology and nomenclature of the by sublytic C5b-9 attack, and the mechanism of ATF3 increasing activating transcription factor/cAMP responsive element binding family of tran- GMCs apoptosis in response to sublytic C5b-9 was associated with scription factors: activating transcription factor proteins and homeostasis. Gene direct up-regulation of Gadd45␣ gene expression and indirect up- 273, 1–11. Huang, X., Li, X., Guo, B., 2008. KLF6 induces apoptosis in prostate cancer cells regulation of KLF6 gene expression. through up-regulation of ATF3. J. Biol. Chem. 283, 29795–29801. Ito, G., Uchiyama, M., Kondo, M., Mori, S., Usami, N., Maeda, O., Kawabe, T., Hasegawa, Y., Shimokata, K., Sekido, Y., 2004. Krüppel-like factor 6 is frequently down- Conflict of interest regulated and induces apoptosis in non-small cell lung cancer cells. Cancer Res. 64, 3838–3843. The authors declare that there is no duality of interest associated James, C.G., Woods, A., Underhill, T.M., Beier, F., 2006. The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and with this manuscript. A gene transcription. BMC Mol. Biol. 7, 30. Jiang, X., Zhang, J., Xia, M., Qiu, W., Wang, H., Zhao, D., Wang, Y., 2010. Role of activating transcription factor 3 (ATF3) in sublytic C5b-9-induced glomerular Acknowledgement mesangial cell apoptosis. Cell. Mol. Immunol. 7, 143–151. Kang, Y., Chen, C.R., Massague, J., 2003. A self-enabling TGF␤ response coupled to This work was supported by grants from National Natural Sci- stress signaling: smad engages stress response factor ATF3 for Id1 repression in epithelial cells. Mol. Cell 11, 915–9126. ence Foundations of China (No. 30772016), Ministry of Education Kawauchi, J., Zhang, C., Nobori, K., Hashimoto, Y., Adachi, M.T., Noda, A., Sunamori, of China (No. 20060312005), Department of Education of Jiangsu M., Kitajima, S., 2002. Transcriptional repressor activating transcription factor Province (No. KJB310004) and Nanjing Medical University (No. 3 protects human umbilical vein endothelial cells from tumor necrosis factor- alpha-induced apoptosis through down-regulation of p53 transcription. J. Biol. 08NMUZ002, 09JC007). We would like to thank Prof. Han Xiao for Chem. 277, 39025–39034. the gift of pTet-off advanced system. Kearsey, J.M., Coates, P.J., Prescott, A.R., Warbrick, E., Hall, P.M., 1995. Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1. Oncogene 11, 1675–1683. Appendix A. Supplementary data Khuu, C.H., Barrozo, R.M., Hai, T., Weinstein, S.L., 2007. Activating transcription factor 3 (ATF3) represses the expression of CCL4 in murine macrophages. Mol. Supplementary data associated with this article can be found, in Immunol. 44, 1598–1605. Kool, J., Hamdi, M., Cornelissen-Steijger, P., van der Eb, A.J., Terleth, C., van Dam, H., the online version, at doi:10.1016/j.imbio.2011.02.005. 2003. Induction of ATF3 by ionizing radiation is mediated via a signaling pathway

that includes ATM nibrin1, stress-induced MAP kinases and ATF-2. Oncogene 22, glomerulonephritis, cell death by a combination of apoptosis and necrosis. 4235–4242. Nephron 86, 152–160. Kojima, S., Hayashi, S., Shimokado, K., Suzuki, Y., Shimada, J., Crippa, M.P., Friedman, Slavin, D., Sapin, V., Lopez-Diaz, F., Jacquemin, P., Koritschoner, N., Dastugue, B., S.L., 2000. Transcriptional activation of urokinase by the Krüppel-like factor Davidson, I., Chatton, B., Bocco, J.L., 1999. The Krüppel-like core promoter bind- Zf9/COPEB activates latent TGF-beta1 in vascular endothelial cells. Blood 95, ing protein gene is primarily expressed in placenta during mouse development. 1309–1316. Biol. Reprod. 61, 1586–1591. Li, D., Yin, X., Zmuda, E.J., Wolford, C.C., Dong, X., White, M.F., Hai, T., 2008. The repres- Tamura, K., Hua, B., Adachi, S., Guney, I., Kawauchi, J., Morioka, M., Tamamori-Adachi, sion of IRS2 gene by ATF3, a stress-inducible gene, contributes to pancreatic M., Tanaka, Y., Nakabeppu, Y., Sunamori, M., Sedivy, J.M., Kitajima, S., 2005. Stress beta-cell apoptosis. Diabetes 57, 635–644. response gene ATF3 is a target of c-myc in serum-induced cell proliferation. Litvak, V., Ramsey, S.A., Rust, A.G., Zak, D.E., Kennedy, K.A., Lampano, A.E., Nykter, M., EMBO J. 24, 2590–2601. Shmulevich, I., Aderem, A., 2009. Function of C/EBPdelta in a regulatory circuit Turchi, L., Fareh, M., Aberdam, E., Kitajima, S., Simpson, F., Wicking, C., Aberdam, D., that discriminates between transient and persistent TLR4-induced signals. Nat. Virolle, T., 2009. ATF3 and p15PAF are novel gatekeepers of genomic integrity Immunol. 10, 437–443. upon UV stress. Cell Death Differ. 16, 728–737. Lu, D., Wolfgang, C.D., Hai, T., 2006. ATF3, a stress-inducible gene, suppresses Ras- Vairapandi, M., Azam, N., Balliet, A.G., Hoffman, B., Liebermann, D.A., 2000. Char- stimulated tumorigenesis. J. Biol. Chem. 281, 10473–10481. acterization of MyD118 Gadd45, and proliferating cell nuclear antigen (PCNA) Maekawa, T., Sano, Y., Shinagawa, T., Rahman, Z., Sakuma, T., Nomura, S., Licht, interacting domains. PCNA impedes MyD118 and Gadd45-mediated negative J.D., Ishii, S., 2008. ATF-2 controls transcription of maspin and GADD45 alpha growth control. J. Biol. Chem. 275, 16810–16819. genes independently from p53 to suppress mammary tumors. Oncogene 27, Wang, Y., He, Q., Qin, H., Xu, J., Tong, J., Gao, L., Xu, J., 2006. The complement C5b- 1045–1054. 9 complexes induced injury of glomerular mesangial cells in rats with Thy-1 Mungrue, I.N., Pagnon, J., Kohannim, O., Gargalovic, P.S., Lusis, A.J., 2009. nephritis by increasing nitric oxide synthesis. Life Sci. 79, 182–192. CHAC1/MGC4504 is a novel proapoptotic component of the unfolded pro- Wang, Q., Mora-Jensen, H., Weniger, M.A., Perez-Galan, P., Wolford, C., Hai, T., Ron, tein response, downstream of the ATF4–ATF3–CHOP cascade. J. Immunol. 182, D., Chen, W., Trenkle, W., Wiestner, A., Ye, Y., 2009. ERAD inhibitors integrate 466–476. ER stress with an epigenetic mechanism to activate BH3-only protein NOXA in Nakagomi, S., Suzuki, Y., Namikawa, K., Kiryu-Seo, S., Kiyama, H., 2003. Expression cancer cells. PNAS 106, 2200–2205. of the activating transcription factor ATF3 prevents c-Jun N-terminal kinase- Warke, V.G., Nambiar, M.P., Krishnan, S., Tenbrock, K., Geller, D.A., Koritschoner, induced neuronal death by promoting heat shock protein 27 expression and akt N.P., Atkins, J.L., Farber, D.L., Tsokos, G.C., 2003. Transcriptional activation of the activation. J. Neurochem. 23, 5187–5196. human inducible nitric-oxide synthase promoter by Krüppel-like factor 6. J. Biol. Perez, S., Vial, E., van Dam, H., Castellazzi, M., 2001. Transcription Chem. 278, 14812–14819. factor ATF3 partially transforms chick embryo fibroblasts by pro- Xu, J.H., Qiu, W., Wang, Y.W., Xu, J., Tong, J.X., Gao, L.J., Xu, W.H., Wu, Y.Q., 2006. The moting growth factor-independent proliferation. Oncogene 20, gene expression profile and overexpression of apoptosis-related genes (NGFI- 1135–1141. B and Gadd45␥) in early phase of Thy-1 nephritis model. Cell Tissue Res. 326, Qiu, W., Che, N., Feng, X., Xia, M., Wang, H., Zhao, D., Wang, Y., 2009. The apoptosis 159–168. of glomerular mesangial cells induced by sublytic C5b-9 complexes in the rats Yamamoto, T., Wilson, C.B., 1987. Complement dependence of antibody induced with Thy-1 nephritis is dependent on up-regulated of Gadd45␥ expression. Eur. mesangial cell injury in the rat. J. Immunol. 138, 3758–3765. J. Immunol. 39, 1–17. Yin, X., Dewille, J.W., Hai, T., 2008. A potential dichotomous role of ATF3, an adaptive- Sánchez, R., Pantoja-Uceda, D., Prieto, J., Diercks, T., Marcaida, M.J., Montoya, G., response gene, in cancer development. Oncogene 27, 2118–2127. Campos-Olivas, R., Blanco, F.J., 2010. Solution structure of human growth arrest Zerbini, L.F., Wang, Y., Correa, R.G., Cho, J.Y., Libermann, T.A., 2005. Blockage of and DNA damage 45alpha (Gadd45alpha) and its interactions with prolifer- NF-kappaB induces serine 15 phosphorylation of mutant p53 by JNK kinase in ating cell nuclear antigen (PCNA) and Aurora A kinase. J. Biol. Chem. 285, prostate cancer cells. Cell Cycle 4, 1247–1253. 22196–22201. Zheng, L., Pan, H., Li, S., Flesken-Nikitin, A., Chen, P.L., Boyer, T.G., Lee, W.H., 2000. Shimizu, A., Masuda, Y., Kitamura, H., Ishizaki, M., Ohashi, R., Sugisaki, Y., Yamanaka, Sequence-specific transcriptional corepressor function for BRCA1 through a N., 2000. Complement-mediated killing of mesangial cells in experimental novel zinc finger protein, ZBRK1. Mol. Cell 6, 757–768.