Báo cáo khoa học: Alpha 1-antichymotrypsin/SerpinA3 is a novel target of orphan nuclear receptor Nur77 potx

Alpha 1-antichymotrypsin/SerpinA3 is a novel target of orphan nuclear receptor Nur77 Yongjuan Zhao, Yanxin Liu and Dexian Zheng State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Orphan nuclear receptor Nur77 (also referred to as TR3 or NGFI-B) is a member of the NR4A family, and is composed of three typical functional domains: an N-terminal region (transactivation function 1, AF-1), a DNA-binding domain, and a ligand-binding domain. Nur77 has no specific ligand discovered hith- erto. The crystal structure of the ligand-binding domain of Nurr1, another member of the subfamily, shows that no cavity is available for binding hydro- phobic side chains of a ligand. This suggests that it might be a real orphan receptor and functional with- out ligand binding [1]. The DNA-binding domain specifically binds to the octanucleotide AAAGGTCA (NBRE) [2] as a monomer, which was found at )65, )1599, )254, )316, )130, )100, )420, )142, and )332 bp upstream to the transcription start sites (TSS) of the genes 21-OHase [3], 20a-HSD [4,5], PAI [6], E2F1 [7], hHSD3B2 [8], INSL3 [9], IKKi [10], GIOT [11], and TCL1 [12], respectively. Nur77 was also reported to activate POMC [13] as a homodimer through another response element, NurRE, which might be regarded as the palindrome of NBRE. However, Keywords gene regulation; genome-wide scan; NBRE; Nur77 transcription factor; SerpinA3 Correspondence D. Zheng, Institute of Basic Medical Sciences, 5 Dong Dan San Tiao, Beijing 100005, China Fax: +86 10 6510 5102 Tel: +86 10 6529 6409 E-mail: zhengdx@pumc.edu.cn or zhengdx@tom.com (Received 17 September 2007, revised 27 December 2007, accepted 3 January 2008) doi:10.1111/j.1742-4658.2008.06269.x Nur77 is one member of the nuclear receptor superfamily. As a transcrip- tion factor, Nur77 participates in a variety of biological processes, includ- ing T cell development, inflammatory responses, steroid hormone synthesis, and hepatic glucose metabolism. It typically acts via binding to the Nur77 responsive element (NBRE) in the promoter regions of its target genes. In the present study, we identified a novel Nur77-regulated gene, a1-anti- chymotrypsin/SerpinA3, via an approach combining computational predic- tion and wet-laboratory validations. First, we identified 483 candidate genes via a human genome-wide scan for NBREs in their proximal pro- moters. Three out of 14 function-associated genes were further identified to be transactivated by Nur77 in luciferase reporter gene assays in HEK 293T cells. The transactivation assay proved that the NBRE ()182 to )175) in the SerpinA3 promoter region is a novel Nur77-dependent functional motif in HEK 293T and HepG2 cells. Electrophoretic mobility shift and chroma- tin immunoprecipitation assays demonstrated that Nur77 physically associ- ates with the SerpinA3 promoter region both in vitro and in vivo. Nur77 overexpression and RNA interference-mediated Nur77 gene knockdown analysis confirmed that SerpinA3 is indeed a novel Nur77-targeted gene. These data may throw light on the function of Nur77 in inflammatory responses and acute-phase reactions as well as the development of Alzheimer’s disease. Abbreviations ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation; DAPI, 4¢,6-diamidino-2-phenylindole; EMSA, electrophoretic mobility shift assay; Ig, immunoglobulin; IL, interleukin; NBRE, NGFI/Nur77 binding responsive element; OSM, oncostatin M; siRNA, small interfering RNA; STAT, signal transducer and activator of transcription; TRITC, tetramethyl rhodamine iso-thiocyanate; TSS, transcription start site. FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS 1025 these known targets cannot fully elucidate the many physiological functions of Nur77, especially in the inflammatory and other immune responses. Thus, we decided that it would be worthwhile to explore the novel target genes of Nur77. In the present study, SerpinA3 (a1-antichymotrypsin) was first identified as a potential Nur77-regulated gene using a genome-wide scan for NBREs in human pro- moter regions. Then, the transcriptional regulation of SerpinA3 by Nur77 was illustrated by in-depth func- tional studies, including transactivation, electrophoretic mobility shift, chromatin immunoprecipitation (ChIP) and RNA interference-mediated gene knockdown. SerpinA3 is a member of the serine protease inhibitor family, and is involved in the inflammatory response. It is mainly synthesized in the liver [14] and is a posi- tive acute-phase reactant. During the acute phase response, the plasma concentration of SerpinA3 increases by almost five-fold. Furthermore, SerpinA3 is implicated in the development of Alzheimer’s disease. It is a major constituent of the plaques, and interacts with the Alzheimer’s neurotoxic amyloid peptide Ab(1- 42) [15]. It is reported that transcriptional factors nuclear factor-jB [16,17], AP-1 [17,18] and signal transducer and activator of transcription-3 (STAT3) [19] regulate SerpinA3 expression in astrocytes upon stimulation with cytokines such as interleukin (IL)-1, oncostatin M (OSM), and tumor necrosis factor-a. In the present study, we report that transcription of SerpinA3 can be regulated by Nur77 via a typical NBRE in its promoter region in HepG2 human liver cancer cells. Results Nur77 response element NBRE in human gene promoters According to data available in the literature [3–12], we assumed that the NBREs in the 5¢-proximal promoters of genes might conduct Nur77-dependent activation signals. To confirm this hypothesis, we downloaded the sequences ()1000 bp to +200 bp relative to the TSS) of all human gene promoters from the DataBase of Transcriptional Start Sites (http://dbtss.hgc.jp), which was constructed based on precise experimentally determined 5¢ end clones [20]. Then we screened for direct and inverted NBREs with the principle of regu- lar expressions in Perl, a programming language with strong text processing abilities, which has become one of the most popular languages for writing bioinformat- ics scripts. We identified 483 candidate genes contain- ing one or two NBREs in their promoter regions (see supplementary Table S1), among which there were 215 direct NBREs (AAAGGTCA) and 286 inverted NBREs (TGACCTTT). Nur77 promotes SerpinA3 promoter activity To determine which genes with NBREs in their promo- ters functionally associated with Nur77, we chose several clusters of genes to construct the promoter-lucif- erase reporter plasmids (Table 1). These gene clusters included steroidogenesis and lipid metabolism [3–5,8], immune responses [13] and inflammatory responses [10], in which Nur77 might have a function. The reporters were then cotransfected into HEK 293T cells with a Nur77 expression plasmid (pcDNA3.1-Nur77) or an empty vector. The relative luciferase activities controlled by the promoters were then detected. As shown in Table 2, three genes, including one known target of Nur77, HSD3B2 [8], and two novel ones, ESR1 and SERPINA3, were identified with relative luciferase activity changing fold ‡ 1.8 (P < 0.01). Unexpectedly, Nur77 failed to induce activation of E2F1, which was in conflict with the report by Mu et al. [7]. ESR1 was set aside because no significant change of ESR1 expression in MCF-7 cells was detected when Nur77 was over- expressed compared to the control (data not shown). Then, we chose the canonically transactivated target, SerpinA3, for further study. Nur77 increases SerpinA3 promoter activity through NBRE ()182 to )175) and/or NBRE-like element ()93 to )88) The SerpinA3 gene is approximately 12.8 kb long, and comprises five exons and four introns. As shown in Fig. 1A,B, in the proximal promoter region of Serpin- A3, there is a TATA-like sequence (TAAATAA) situ- ated at positions )16 to ) 9 relative to the annotated TSS. Two STAT binding sites are located at positions )112 to )105 (STAT A) and )82 to )73 (STAT B), which are the STAT3 binding sites in cytokine OSM- stimulated astrocytes [19]. The recognition sequence of Nur77 appears to be conserved during evolution, although the distance to each TSS is different. NBREs were found in human SerpinA3 (TGACCTTT, positions )81 to )175 from TSS), mouse SerpinA3n (AAAGGTCA, positions )1138 to )1131 from TSS), and rat SerpinA3n (TA AGGTCA, positions )1265 to )1258 from TSS) (Fig. 1C). The SerpinA3n of mouse and rat are the closest orthologues of human SerpinA3 [21,22]. This suggested that NBREs might be important to the regu- lation of SerpinA3 expression. SerpinA3 is a novel target of Nur77 Y. Zhao et al. 1026 FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS To confirm the activation of SerpinA3 promoter by Nur77, cell lines including HEK 293T, hepatocytes [HepG2, SMMC-7721 and primary human hepatic (PHH)], and prostate cancer cells (LNCaP and PC-3) were cotransfected with the Nur77 expression plasmid and the SerpinA3-luciferase reporter plasmid, pGL3- Ser. Nur77-dependent luciferase activity assays showed that Nur77 activated the SerpinA3 promoter with vari- ous degrees in most cell lines (P £ 0.01 or P £ 0.05) (Fig. 2A), except PHH and PC-3 (P > 0.05). Table 2. Luciferase activity driven by promoters of candidate genes. pGL3-basic-X: the gene symbols of which promoters were cloned into plasmid pGL3-basic. –, the empty vector; NBRE, NBRE-tk-Luc; pcDNA3.1 and pcDNA3.1-Nur77, relative luciferase activity of the correspond- ing constructs, which were measured after 48 h of cotransfection with pcDNA3.1 and pcDNA3.1-Nur77 plasmid, respectively. The firefly luciferase activity was normalized to renilla luciferase activity. The data shown are from a representative experiment reported as the mean ± SD (n = 3). Ratio, ratio of the mean activity of the construct cotransfected with pcDNA3.1-Nur77 to that with pcDNA3.1. pGL3-basic-X pcDNA3.1 pcDNA3.1-Nur77 Ratio P C4B 1.442 ± 0.286 1.043 ± 0.078 0.723 0.079 CCL20 0.026 ± 0.006 0.030 ± 0.008 1.154 0.566 COVA1 6.023 ± 0.279 5.205 ± 0.747 0.864 0.150 CYP21A2 0.072 ± 0.004 0.057 ± 0.005 0.792 0.140 CYP39A1 0.386 ± 0.011 0.407 ± 0.050 1.054 0.515 E2F1 9.562 ± 0.939 9.559 ± 2.060 1.000 0.504 ESR1 0.219 ± 0.042 0.513 ± 0.044 2.342 0.001** FLAD1 0.920 ± 0.051 1.110 ± 0.037 1.207 0.006** HSD3B2 0.046 ± 0.005 0.083 ± 0.009 1.804 0.004** IL1B 0.417 ± 0.040 0.265 ± 0.065 0.635 0.026 MYL7 0.112 ± 0.004 0.166 ± 0.044 1.482 0.104 NDUFS1 25.792 ± 2.631 15.185 ± 0.546 0.589 0.002** SERPINA3 0.370 ± 0.033 2.548 ± 0.175 6.886 2.96 · 10 )5 ** UQCRH 6.256 ± 0.134 7.822 ± 1.009 1.250 0.471 – 0.046 ± 0.000 0.044 ± 0.000 0.957 0.866 NBRE 0.432 ± 0.061 4.872 ± 0.478 11.278 0.006** ** P £ 0.01 (one-way ANOVA). Table 1. Information of candidate genes with NBRE in the promoter regions. NBRE position, the position of the first and the last base of NBRE to TSS; direction: +, AAAGGTCA; ), TGACCTTT (both in the sense strand of the promoters). Gene symbol RefSeq ID Gene definition NBRE position Direction C4B NM_000592 Homo sapiens complement component 4B )871/)864 + CCL20 NM_004591 Homo sapiens chemokine (C-C motif) ligand 20 )335/)328 ) COVA1 NM_006375 Cytosolic ovarian carcinoma antigen 1 )882/)875 + CYP21A2 NM_000500 Homo sapiens cytochrome P450, family 21, subfamily A, polypeptide 2 )235/)228 + CYP39A1 NM_016593 Homo sapiens cytochrome P450, family 39, subfamily A, polypeptide 1 )828/)821 + E2F1 NM_005225 Homo sapiens E2F transcription factor 1 )333/)326 + ESR1 NM_000125 Estrogen receptor 1 )813/)806 + FLAD1 NM_025207 Flavin adenine dinucleotide synthetase, homolog (yeast) )502/)495 + HSD3B2 NM_000198 Homo sapiens hydroxy-delta-5-steroid dehydrogenase, 3b- and steroid delta-isomerase 2 )131/)124 + IL1B NM_000576 Homo sapiens IL-1b )559/)552 + MYL7 NM_021223 Homo sapiens myosin, light polypeptide 7, regulatory )462/)455 + NDUFS1 NM_005006 Homo sapiens NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa (NADH-coenzyme Q reductase), nuclear gene encoding mitochondrial protein, )328/)321 + SERPINA3 NM_001085 Serpin peptidase inhibitor, clade A (a-1 antiproteinase, antitrypsin), member 3 )182/ )175 ) UQCRH NM_006004 Homo sapiens ubiquinol-cytochrome c reductase hinge protein )324/)317 + Y. Zhao et al. SerpinA3 is a novel target of Nur77 FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS 1027 To determine whether the up-regulation of SerpinA3 gene promoter activity by Nur77 was through its NBRE ()182 to )175), the NBRE was deleted from the pGL3-Ser reporter plasmid to generate pGL3-St- 182 (Fig. 2B). Transient cotransfection of HEK 293T cells with pGL3-St-182 and pcDNA3.1-Nur77 showed that Nur77 up-regulated SerpinA3 promoter activities in a dose-dependent manner (Fig. 2C, columns 2–4 versus column 1) and deletion of the NBRE ()182 to )175) weakened the transactivation significantly A B C Fig. 1. Structure and nucleotide sequences of the proximal promoter region of human SerpinA3. (A) Schematic presentation of the 5¢ Serpin- A3 promoter region. The predicted transcription start site is indicated as ‘+1’ [20]. Transcriptional regulatory elements present in the Serpin- A3 promoter region are shown, which included the TATA box, two known functional elements (STAT A and STAT B) [19], one NBRE and two NBRE-like elements, which contained the core response sequence of nuclear receptors. Numbers indicate the positions relative to the TSS (bent arrow). (B) A nucleotide sequence alignment of the human SerpinA3 5¢-proximal promoter is presented with cis-regulatory ele- ments of STAT A (from )112 to )105), STAT B (from )82 to ) 73), Nur77 (putative, from )595 to )590, from )182 to )175 and from )93 to )88) or TATA box (from )16 to )9) (boxed). (C) Comparison of the nucleotide sequences of an authentic NBRE consensus sequence (AAAGGTCA) versus the putative NBRE elements present in human SerpinA3 (TGACCTTT, from )182 to )175), mouse SerpinA3n (AAAGGTCA, from )1138 to )1131), and rat SerpinA3n (TAAGGTCA, from )1265 to )1258). Numbers indicate nucleotide distances from the default TSS. SerpinA3 is a novel target of Nur77 Y. Zhao et al. 1028 FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS (Fig. 2C, columns 6–8 versus columns 2–4). Thus, the NBRE ()182 to )175) is a necessary element for the transactivation of SerpinA3 by Nur77. Further sequence analysis showed that there were two NBRE-like elements in the proximal promoter region of SerpinA3 (Fig. 1B). Compared with the intact NBRE, there was a difference in one or two of the flank base pairs. We assumed that these two NBRE-like elements might also contribute to the Nur77-mediated SerpinA3 transactivation. To confirm this hypothesis, four additional reporter plasmids, pGL3-St-93, pGL3-St-93/-182, pGL3-Lser, and pGL3- Lst-595 with various deletions were constructed (Fig. 2B). Promoter transactivity assays showed that the transactivation of SerpinA3 by Nur77 in HEK 293T cells was weakened by deleting NBRE-like ele- ment ()93 to )88) only (Fig. 2C, lanes 9–12 versus lanes 1–4) and completely eliminated when NBRE ()182 to )175) and NBRE-like element ()93 to )88) were both deleted (Fig. 2C, columns 13–16 versus col- umns 1–4), indicating that the NBRE-like element ()93 to )88) might also be a functional element in HEK 293T cells. However, deletion of the NBRE ()182 to )175) led to complete elimination of Serpin- A3 activation by Nur77 and deletion of the NBRE-like element ()93 to )88) had no significant effect in HepG2 cells (Fig. 2D). This suggests that the function of the NBRE-like element ()93 to )88) might be cell line dependent. The deletion of another NBRE-like element ()595 to )590) did not impair the activation of the SerpinA3 promoter ()700 to +54) by Nur77 (Fig. 2C, columns 21–24 versus columns 17–20). Altogether, these data demonstrated that Nur77 up- regulated SerpinA3 promoter activity mainly by the NBRE ()182 to )175) in HepG2 cells and the NBRE- like element ()93 to )88) also contributed to SerpinA3 activation in HEK 293T cells. Nur77 protein binds to NBRE in SerpinA3 promoter in vitro We further assessed DNA binding specificity of Nur77 to the NBRE ()182 to )175) and NBRE-like elements 0 2 4 6 8 10 12 14 A B C D HEK 293T SMMC- 7721 PC-3 LNCaPHepG2PHH Relative Luc activity vector Nur77 ** ** ** * +54 Luc Luc Luc Luc Luc Luc –700 –595 –590 –341 –182–175 –93–88 Ser St-182 St-93 St-182/93 Lser Lst-595 6 5 4 3 2 1 0 Relative Luc activity vector Nur77 Nur77 Ser 1 2 3 4 5 6 7 8 9101112 13141516 17181920 21222324 St-182 St-93 St-182/93 Lser Lst-595 0 3 6 9 12 15 18 21 Relative Luc activity vector Nur77 Ser St-182 St-93 St-182/93 Lser Lst-595 Fig. 2. Activation of the SerpinA3 promoter by Nur77 in the cell lines. (A) HEK 293T, hepatocytes (HepG2, SMMC-7721 and PHH) and prostate cancer cells (LNCaP and PC-3) were cotransfected with SerpinA3 luciferase reporter plasmid pGL3-Ser ()341 to +54) and either pcDNA3.1-Nur77 expression plasmid ( ) or the empty vector (h). Cells were lysed with lysis buffer and the lysates were subjected to luciferase activity assay. **P £ 0.01; *P £ 0.05, one- way ANOVA. (B) A diagram of various NBRE deletions in the Serp- inA3 promoter Luc reporter genes used in transient transfections. (C) Deletions of the putative NBRE ()182 to )175) and the NBRE- like element ()82 to )73) in SerpinA3 promoter significantly reduced or abolished SerpinA3 promoter activity induced by Nur77 in HEK 293T cells. SerpinA3 promoter reporter constructs in (B) were cotransfected with an empty expression vector (vector; h)or increasing amounts (25, 50, and 100 ng) of pcDNA3.1-Nur77 plas- mids ( ) into HEK 293T cells. Measurement of luciferase activity and data analysis were conducted as in Table 2. (D) Deletions of NBRE ()182 to )175) abolished SerpinA3 promoter activation induced by Nur77 in HepG2 cells. SerpinA3 promoter reporter con- structs in (B) were cotransfected with an empty expression vector (vector; h) or pcDNA3.1-Nur77 ( ) into HepG2 cells. Measurement of luciferase activity and data analysis were conducted as in (C). Y. Zhao et al. SerpinA3 is a novel target of Nur77 FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS 1029 by electrophoretic mobility shift assay (EMSA). As shown in Fig. 3, in vitro translated Nur77 protein (ver- ified by western blot, Fig. 3A) bound to the NBRE ()182 to )175) with high affinity (Fig. 3B, lanes 1–2). It did not bind to the other two NBRE-like elements, with or without anti-Nur77 pre-incubation (Fig. 3B, lanes 15–20). Nur77 binding was specifically competed by increasing the concentrations (2.5- to 50-fold) of unlabeled oligonucleotides (Fig. 3B, lanes 3–5), but not by oligonucleotides harboring a mutation or deletion in the NBRE element (Fig 3B, lanes 6–11) or nonspe- cific oligonucleotides (Fig. 3B, lane 12). This result is consistent with the specific requirement for the NBRE ()182 to )175) in Nur77-mediated activation of the SerpinA3 promoter (Fig. 2C,D). To further determine whether Nur77 was present in the DNA-protein complex, the reactant was pre-incu- bated with an antibody against Nur77. As shown in Fig. 3B (lanes 13 and 14), addition of antibody against Nur77 prevented the formation of a Nur77-DNA com- plex and produced a slight supershift band above the former position, whereas anti-Sp1 (serving as a nonspe- cific antibody) had no such effect on the band shift. Taken together, these data demonstrate that the Nur77 protein specifically binds to the NBRE in the SerpinA3 promoter in vitro. Nur77 protein binds to the SerpinA3 promoter in HepG2 cells (in vivo) To determine whether Nur77 actually binds to the SerpinA3 promoter in vivo, ChIP assays were performed in the human hepatoma cell lines HepG2 and SMMC-7721 and the glioma cell line U251. As shown in Fig. 4, a fragment from )341 to +54 of the SerpinA3 promoter containing the NBRE ()182 to )175) was amplified from the anti-Nur77 chromatin immunoprecipitant by PCR, but a nonspecific anti- body (rabbit IgG) did not precipitate the promoter sequence. Thus, Nur77 was recruited to the promoter region of SerpinA3 in HepG2 and U251, but not in SMMC-7721 cells (Fig. 4B). This is consistent with the fact that SerpinA3 is expressed in both HepG2 and U251, but not in SMMC-7721 cells (Fig. 4A). These results suggest that the Nur77 protein binds to the active promoter of SerpinA3 in vivo. Additionally, ChIP assays were performed in a stable HepG2 cell line expressing a GST-Nur77 fusion protein by using antibodies against Nur77 or GST. As shown in Fig. 4C, the SerpinA3 promoter was activated by the GST-Nur77 fusion protein, and both anti-Nur77 and anti-GST sera precipitated the promoter fragment ()341 to +54) of SerpinA3 (Fig. 4D). This confirmed that Nur77 associates specifically with the SerpinA3 pro- moter containing the NBRE ()182 to )175) in vivo. Nur77 enhances endogenous SerpinA3 expression in HepG2 cells To determine whether Nur77 could enhance endo- genous SerpinA3 mRNA levels, HepG2 cells were transfected with Nur77 expression plasmid (pcDNA3.1- Nur77) or the empty vector pcDNA3.1, and then Serp- inA3 transcription was measured by semi-quantitative PCR and real-time quantitative PCR and the expression of Nur77 was confirmed by western blot. As shown in Fig. 5A, the SerpinA3 transcript level measured by semi-quantitative PCR was enhanced in Nur77 expres- sion plasmid-transfected cells significantly compared to cells transfected with the empty vector. The real-time PCR revealed a significant 3.98-fold difference in A B Fig. 3. NBRE element ()182 to )175) was a Nur77-specific binding site. (A) In vitro expression of Nur77 was confirmed by western blot. (B) EMSA was used to assess the binding of in vitro produced Nur77 to the double-stranded 32 P-labeled oligonucleotides corre- sponding to the three putative Nur77 response elements [NBRE- like element ()595 to )590), NBRE ()182 to )175) and NBRE-like element ()93 to )88)], referred to as Probe-595, Probe-182, and Probe-93, respectively. Binding of Nur77 was then challenged by increasing doses (D, molar excesses of 2.5-, five-, and 50-fold) of unlabeled oligonucleotides corresponding to the wild-type )182/ )175 element (wt-182), the )182/)175 element mutated (mut- 182), the )182/)175 element truncated (trunc-182) and the nonspe- cific oligonucleotide (n.s.). Nur77 binding was supershifted by a Nur77 antiserum (aNur77) or nonspecific antibody (n.s., aSp1). There was no specific binding of Nur77 found with either Probe-93 or Probe-595. SerpinA3 is a novel target of Nur77 Y. Zhao et al. 1030 FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS SerpinA3 mRNA levels between Nur77 expression plas- mid- and empty vector-transfected cells (Fig. 5B). Furthermore, immunofluorescence microscopy dem- onstrated that there was a higher level of SerpinA3 translation in EGFP-Nur77 positive HepG2 cells tran- siently transfected with pEGFP-Nur77 expression plas- mid (Fig. 5C,D), indicating that Nur77 overexpression enhances endogenous SerpinA3 expression in HepG2 cells. Nur77-specific small interfering RNA (siRNA) attenuates endogenous SerpinA3 expression in HepG2 cells Finally, the effect of down-regulation of Nur77 by RNA interference on endogenous SerpinA3 expression was investigated in HepG2 cells transfected with Nur77-specific siRNA or nontargeting siRNA as a control. Nur77 and SerpinA3 expression was measured by semi-quantitative RT-PCR, real-time PCR, or western blot assays. As shown in Fig. 6A, down-regu- lation of Nur77 by siRNA resulted in a reduction of SerpinA3 mRNA content in the HepG2 cells; the level of endogenous SerpinA3 mRNA in Nur77-siRNA- transfected cells was reduced by 75% (Fig. 6B). Western blot assays (Fig. 6C) showed that down- regulation of Nur77 protein by siRNA resulted in the reduction of SerpinA3 proteins in HepG2 cells. Serpin- A3 consists of three cellular SerpinA3 with molecular weights of 37, 46, and 62 kDa; these might be the results of glycosylation events [22]. In addition, there was one secretory version of SerpinA3 with a molecu- lar weight of 62 kDa. Thus, down-regulation of Nur77 attenuated SerpinA3 expression at both the mRNA and protein levels in HepG2 cells. Discussion Nur77 is an early response gene, which can be induced by various signals such as fatty acids [23], AB CD Fig. 4. Nur77 bound to human SerpinA3 promoter in vivo. (A) RT-PCR analysis of SerpinA3 transcript levels in three cell lines: HepG2, U251 and SMMC-7721. Cells were harvested during the exponential growth phase and RNAs were extracted with TRIzol reagent. Two lg of RNAs were reverse-transcribed and cDNAs were subjected to PCR using SerpinA3-specific primer pairs: ACTas5-ACTas3 and ACT5-ACT3. With the primer pair ACTas5-ACTas3, the specific product was 1.1 kb in length and the smaller band was nonspecific as determined by DNA sequenc- ing. (B) ChIP was performed on the extracts of HepG2, U251 and SMMC-7721 cells as described in the Experimental procedures. Immuno- precipitation was performed by using a specific anti-Nur77 serum or rabbit IgG (control) and primers spanning the SerpinA3 promoter region ()341 to +54) were used for PCR amplification. Templates containing 10%, 1%, and 0.2% of the pre-immunoprecipitation input genomic DNAs were amplified as controls. (C) The transactivity of the fusion protein GST-Nur77 to SerpinA3 promoter was confirmed by luciferase assay. (D) ChIP assays were carried out on extracts from a stably transfected HepG2 cell line with pcDNA3.1-GST-Nur77. Immunoprecipitation was performed using antibodies against GST and Nur77, or rabbit IgG (control) and PCR was performed as described above. Y. Zhao et al. SerpinA3 is a novel target of Nur77 FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS 1031 stress [24], prostaglandins [25], growth factors [26], calcium [27], inflammatory cytokines [10,28], peptide hormones [29], phorbol esters [27], and neurotrans- mitters [30]. In addition to playing a role in the mito- chondrial pathway induced by apoptotic stimuli [31], Nur77 is thought to exert its effects by acting as a AB C D Fig. 5. Effects of Nur77 on endogenous SerpinA3 transcript and expression levels in HepG2 cells. (A) Semiquantitative RT-PCR analysis of SerpinA3 transcript levels. HepG2 cells were transfected with 4 lg of Nur77 expression plasmid pcDNA3.1-Nur77 or empty vector pcDNA3.1 for 48 h in the 35 mm wells. Total protein and RNA were extracted from equal amounts of HepG2 cells. The protein was used in a western blot assay for Nur77 quantification, whereas the RNA was reverse-transcribed and cDNA was subjected to PCR using gene-spe- cific primers for SerpinA3 and b-actin. b-actin was used for normalization. (B) Relative SerpinA3 transcript levels quantified by realtime quanti- tative RT-PCR. Representative results were shown from one of three experiments, which were performed using different preparations of total RNA. The SerpinA3 transcript level is significantly (n =3,P < 0.01) different from vector control. (C) The transactivity of the fusion pro- tein EGFP-Nur77 to SerpinA3 promoter was confirmed by luciferase assay. (D) Immunofluorescence study of HepG2 cells transiently trans- fected with pEGFP-Nur77. EGFP-Nur77 protein expression is shown in green, SerpinA3 was labeled with a primary mAb and a secondary TRITC-conjugated antibody (red), and nuclei were stained with DAPI (blue). SerpinA3 is a novel target of Nur77 Y. Zhao et al. 1032 FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS transcription factor. Its target genes still remain lar- gely unexplored except for those genes mentioned above [3–12]. Identification of transcription factor-target genes is a vital step towards understanding the regulatory mecha- nism of gene expression. ChIP-chip, which combines the techniques of ChIP and microarray hybridization, is a popular method to explore target genes of tran- scription factors. For a transcription factor with a known binding motif, genome-wide sequence searches of promoter regions have been applied to predict the target genes [32–34]. We were motivated to identify the novel target genes of Nur77, whose response element was well known. Based on the assumption that the genes with NBREs in their promoters may potentially be regulated by Nur77, we utilized in silico scanning to screen the potential target genes. The promoters of 483 genes have been shown to contain one or two NBREs. We checked 14 function-associated genes and found that only two genes could actually be transactivated by Nur77, including one known target gene, HSD3B2, and one novel potential target, SerpinA3. This suggests that NBREs presenting in promoter regions might be necessary but not sufficient for gene transactivation by Nur77. It might be interesting to investigate why some NBRE-containing genes could be activated by Nur77 whereas others could not. We further present several lines of evidence showing that SerpinA3 was a novel target gene of Nur77. First, the expression of SerpinA3 was regulated by Nur77 in HepG2 cells. When Nur77 was overexpressed, both mRNA and protein levels of SerpinA3 were increased; when the expression of Nur77 was suppressed, the expression of SerpinA3 was reduced. Second, analyses of the regulatory elements of SerpinA3 demonstrated that the NBRE ()182 to )175) was crucial for its expression in HepG2 cells and another NBRE-like ele- ment ()93 to )88) contributed to its Nur77-dependent transactivation in HEK 293T cells. Third, gel mobility shift and ChIP assays demonstrated direct binding of Nur77 to the SerpinA3 proximal promoter both in vitro and in vivo. Recently, 12 DNase I-hypersensitive sites were iden- tified distributed throughout the entire subcluster of SerpinA3 in HepG2 cells [35], providing further evi- dence to our discovery. The NBRE ()182 to )175) motif in our study was located in DNase I-hypersensi- tive site 6 [35], which furnished the accessibility to the SerpinA3 promoter for Nur77. Taken together, these data suggest that Nur77 is an important factor regulat- ing the expression of SerpinA3. Nur77 is reported to be involved in the inflamma- tory response [10,28,36]. Our data might enrich the evidence of this discovery and provide one possible mechanism. The study of the transcriptional regulation of human SerpinA3 was focused on cytokines such as OSM [19], IL-1 [16,18], tumor necrosis factor-a [17], and IL-6/soluble IL-6 receptor [19] stimulated HDPAG 77ruN A B C C o n rt o l N u r 7 7 s i R N A 3 An i pr e S βnitca- 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 ANR i s77 r u Nl or tn oC Relative SerpinA3 transcript level )52.0 ( * 77ruN 3Anip re Sc 3A ni preSs H D PAG Dk26 D k64 Dk73 Dk26 77 ru Nh slo r tno C Fig. 6. Effects of Nur77-specific siRNA on endogenous SerpinA3 gene expression in HepG2 cells. (A, B) Semiquantitative RT-PCR and real-time PCR analysis of SerpinA3 transcript levels. HepG2 cells were transfected with 90 nmol Nur77-specific siRNA or con- trol siRNA (control) for 48 h in the 35 mm wells. Total protein and RNA were extracted from equal amounts of HepG2 cells. The quan- titative experiments were performed as shown in Fig. 5A,B. (C) Western blot analysis of Nur77 and SerpinA3 protein levels. HepG2 cells were transfected with pSilencer-shNur77 and empty vector (control) by Lipofectamine 2000. 24 h later, the cells were applied to nonserum culture media. After another 24 h, culture supernatant and cell lysates were collected for western blot analysis. Y. Zhao et al. SerpinA3 is a novel target of Nur77 FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS 1033 astrocytes, partly because of their involvement in the generation of Alzheimer’s disease. The transcription factors involved in these processes included nuclear factor-jB [16,17], AP-1 [18], and STAT3 [19]. Our unpublished data show that Nur77 regulates SerpinA3 in the human glioma U251 cell line in the same way as in HepG2 cells, although the expression level of Serp- inA3 was much lower in U251 than that in HepG2. This suggests that Nur77 might be a novel trans-acting factor contributing in this pathological process. The biological functions of this transactivation event will be the subject of further study. In the present study, we found that another NBRE- like element ()93 to )88) might also contribute to the Nur77-dependent SerpinA3 activation in HEK 293T cells, although strong binding was not observed by in vitro translated-Nur77 protein in EMSA (Fig. 3B). The physical association between Nur77 and STATs [37] provided one possible mechanism: Nur77 might function via the NBRE-like element ()93 to )88) with the help of STATs. Experimental procedures Bioinformatic analysis Human promoter sequences were downloaded from the DataBase of Transcriptional Start Sites [20], which covered ()1000, +200) segments relative to each default TSS. The dataset used comprised Version 4.0 (released July 2004). A search of direct and inverted NBRE (AAAGGTCA) sites was performed with the principle of regular expressions in Perl. Antibodies and chemicals Polyclonal antibodies against Nur77 (sc-5569) and Sp1 (sc-14027) were purchased from Santa Cruz Biotechnologies Inc. (Santa cruz, CA, USA). Monoclonal antibody against human SerpinA3/a1-antichymotrypsin (AF1295) was pur- chased from R&D Systems Inc. (Minneapolis, MN, USA). Monoclonal antibody against GAPDH was purchased from Kangchen Bio-tech (Shanghai, China). The peroxidase- and tetramethyl rhodamine iso-thiocyanate (TRITC)-conjugated anti-mouse or anti-rabbit IgG sera were obtained from Zhongshan Biotechnology Co. (Beijing, China). 4¢,6-diami- dino-2-phenylindole (DAPI) was purchased from Sigma (St Louis, MO, USA). Cell culture HEK 293T and PHH cells were cultured in DMEM with complement of 10% fetal bovine serum (Hyclone, Logan, UT, USA). SMMC-7721, U251, LNCaP, and PC-3 were cultured and maintained in RPMI 1640 medium with 10% fetal bovine serum. Human hepatoma cell line HepG2 was cultured and maintained in Earle’s minimum- essential medium with non-essential amino acids with 10% fetal bovine serum. All the cells were incubated at 37 °Cin5%CO 2 . Plasmid constructs The promoters of gene X (X: C4B, CCL20, COVA1, CYP21A2, CYP39A1, E2F1, ESR1, FLAD1, HSD3B2, IL1B, MYL7, NDUFS1, SERPINA3, UQCRH) were ampli- fied by PCR from SMMC-7721 genomic DNA and then inserted into pGL3-basic vector to generate pGL3-basic- X plasmids. Construction information, including primers, restriction sites, and product length, is provided in the sup- plementary Table S2. To generate NBRE-truncated Serpin- A3 promoters, large primer PCR was performed with primers harboring the mutations, 5¢-CTAATCTCTT CCTCCAAAAAGCACACAGA-3¢ for St-182 and St-93/ -182, 5¢-AGAAATTATCATCTTTTCCAGTCCGAGA-3¢ for St-93 and St-93/-182, and 5¢-TGGTCTTGAACTCCT CGTGATCTGCCCA-3¢ for Lst-595. pcDNA3.1-Nur77 expression plasmid was a gift from H. Harant [38]. NBRE-tk-Luc reporter construct was kindly provided by H. S. Choi [39]. pEGFP-Nur77 was constructed by inserting SacI-ApaI fragment of Nur77 into the pEGFP- C1 in the laboratory. pcDNA3.1-GST was constructed by inserting the fragment generated by PCR with primers 5¢-AAGGaagcttGCCACCatgtcccctatacta-3¢ (with Hind III, Kozak sequence and a translation start coden), 5¢- aattGGGCCCtcagtcagtcacgatgc-3¢(with ApaI) from pGEX- 4T-3 (Amersham Pharmacia, Piscataway, NJ, USA), then a BamHI-XhoI fragment of Nur77 PCR product was inserted into pcDNA3.1-GST to generate pcDNA3.1-GST-Nur77 plasmid. pSilencer-shNur77 was prepared by overlapping strategy with primers 5¢-gacGGATCCgcagtccagccatgctccttt caagagaaggagcatg-3¢ (with BamHI), 5¢-cggAAGCTTtATC GATccaaaaaacagtccagccatgctccttctcttg-3¢ (with HindIII and ClaI) with 16 complementary bases, which were denatured, annealed and extended with DNA polymerase (Tiangen, Beijing, China), then inserted into pSilencer 3.1-H1 (Ambion, Austin, TX, USA) after BamHI-HindIII digestion. All the fragments inserted into the plasmids were confirmed by DNA sequencing by Beijing Nuosai Gene Company (Beijing, China). Transient transfection and luciferase assay Cells were transiently transfected with plasmids by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, 2 · 10 4 HEK 293T cells per well in 96-well plates were SerpinA3 is a novel target of Nur77 Y. Zhao et al. 1034 FEBS Journal 275 (2008) 1025–1038 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... gels, and visualized by autoradiography Oligonucleotides (sense strand) used for EMSA were probe-93, 5¢-GAAATTATCATCTGGTCATTTCCAGTCC G-3¢, truncated probe-93, 5¢-GAAATTATCATCTTTTCC AGTCCG-3¢, probe-182, 5¢-GTGTGCTTTTTTGACCTTT GGAGGAAGAG-3¢, truncated probe-182, 5¢-GTGTGCT TTTTGGAGGAAGAG-3¢, mutant probe-182, 5¢-GTGT GCTTTTTTGTTCTTTGGAGGAAGAG-3¢, probe-595, 5¢-GCAGATCACGAGGTCAGGAGTTCAAGAC-3¢, truncated probe-595,... CGCAGCTCATTGTAGAAGG-3¢ Another primer pair used for SerpinA3 was ACTas5-ACTas3: 5¢-GAATCC ACCAGCTACATCCA-3¢ and 5¢-GTGCCCTCCTCAAA TACATCA-3¢ Western blot assay The cells were homogenized in lysis buffer (1 · NaCl/Pi, 1% NP40, 13.7 lgÆmL)1 pepstatin A, 0.7 mm EDTA and protease inhibitor cocktail tablets; Roche, Mannheim, Germany) The lysates were subjected to SDS/PAGE and western blot analysis with... Statistical analysis Data are expressed as the mean ± SD (n = 3), with significant differences assessed using one-way analysis of variance (ANOVA) at P < 0.05 or P < 0.01 9 Acknowledgements We thank Dr Hanna Harant (Novartis Institute for BioMedical Research, Austria) for the plasmid pcDNA3.1 -Nur77, Mr Zhijun Yu for help with program writing, Dr Ming Li for pEGFP -Nur77, Dr Feng Yang for technical assistance... min, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min Each reaction was performed in triplicate The data were quantified using sequence detection Software version 1.2 (Applied Biosystems) The primer pair for SerpinA3 was ACT5-ACT3: 5¢-GACTCGCAGACAATGATGG TC-3¢ and 5¢-GCAAACTCATCATGGGCACC-3¢ The results were normalized with b-actin, for which the primers were 5¢-ATGGTGGGAATGGGTCAGAAG-3¢ and 5¢-CA CGCAGCTCATTGTAGAAGG-3¢... Tsujimoto G, Matsumoto K, Saito H & Hashida R (2005) Prostaglandin A2 acts as a transactivator for NOR1 (NR 4A3 ) within the nuclear receptor superfamily Biol Pharm Bull 28, 1603–1607 SerpinA3 is a novel target of Nur77 26 Hazel TG, Nathans D & Lau LF (1988) A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily Proc Natl Acad Sci USA 85, 8444–8448... specific antibodies against Nur77, SerpinA3, and GAPDH, respectively, followed by horseradish peroxidase coupled secondary antibodies and visualized using ImmobilonÔ western blot reagents (Millipore Corporation, Billerica, MA, USA) and exposured to X-ray film RNA interference assay Nur77- specific siRNA (5¢-CAGUCCAGCCAUGCUC CUtt-3¢) was designed according to a previous study [40], SerpinA3 is a novel target of. .. supplementary material is available online: Table S1 Information of 483 candidate genes with NBRE in the promoter regions Table S2 Cloning information of pGL3-basic-X This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any... SerpinA3 is a novel target of Nur77 Y Zhao et al again, and incubated for 12 min in 0.5% Triton X-100/ NaCl/Pi After three more washes, the cells were incubated in 3% BSA/NaCl/Pi for 30 min at 37 °C, then transferred to antibody against SerpinA3 (1 : 50 dilution in NaCl/Pi) containing 3% BSA at 4 °C overnight, washed abundantly in NaCl/Pi, and incubated for 1 h with the TRITC-conjugated secondary antibody... assistance and Dr Dapeng Zhang for help with preparation of the manuscript This work was partially supported by Natural Science Foundation of China Grant Numbers 30571687 and 30640097, and State Key Basic Research Program of China (973) Grant No 2007CB507404 10 11 12 References 1 Wang Z, Benoit G, Liu J, Prasad S, Aarnisalo P, Liu X, Xu H, Walker NP & Perlmann T (2003) Structure and function of Nurr1... min at 30 °C, the cells were washed and processed for ChIP assay as described previously [11] Anti -Nur77 serum was used for immunoprecipitation Immunoprecipitated DNA and input sheared DNA were subjected to PCR using SerpinA3 primer pairs: 5¢-GGTACCACCAAAATGTT AGCTGGG-3¢ and 5¢-AAGCTTGATGTAGCTGGTGGA TTC-3¢, which amplified a 395-bp fragment ()341 to +54) containing the NBRE ()182 to )175) of SerpinA3 . Alpha 1-antichymotrypsin/SerpinA3 is a novel target of orphan nuclear receptor Nur77 Yongjuan Zhao, Yanxin Liu and Dexian Zheng State Key Laboratory of. mutations, 5¢-CTAATCTCTT CCTCCAAAAAGCACACAGA-3¢ for St-182 and St-93/ -182, 5¢-AGAAATTATCATCTTTTCCAGTCCGAGA-3¢ for St-93 and St-93/-182, and 5¢-TGGTCTTGAACTCCT CGTGATCTGCCCA-3¢