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.. In the present study,
Trang 1orphan 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 validapredic-tions 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.
Trang 2these 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
Trang 3To 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.
** 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
NBRE position Direction
CYP21A2 NM_000500 Homo sapiens cytochrome P450, family 21, subfamily A,
polypeptide 2
CYP39A1 NM_016593 Homo sapiens cytochrome P450, family 39, subfamily A,
polypeptide 1
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
NDUFS1 NM_005006 Homo sapiens NADH dehydrogenase (ubiquinone) Fe-S
protein 1, 75 kDa (NADH-coenzyme Q reductase), nuclear gene encoding mitochondrial protein,
SERPINA3 NM_001085 Serpin peptidase inhibitor, clade A (a-1 antiproteinase, antitrypsin),
member 3
UQCRH NM_006004 Homo sapiens ubiquinol-cytochrome c reductase hinge protein )324/)317 +
Trang 4To 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¢ 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.
Trang 5(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,
St-93, St-93/-182, 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-A3activation 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 LNCaP HepG2 PHH
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
Nur77 Ser
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
St-182 St-93 St-182/93 Lser Lst-595
0 3 6 9 12 15 18 21
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).
Trang 6by 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
SerpinA3promoter (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 SerpinA3promoter 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 32P-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.
Trang 7SerpinA3mRNA 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 SerpinA3mRNA 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 SerpinSerpin-A3 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],
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.
Trang 8stress [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
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 Represenquanti-tative 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).
Trang 9transcription 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 trantran-scription 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 vitroand 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
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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 (concon-trol) 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.
Trang 10astrocytes, 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-minimum-essential amino acids with 10% fetal bovine serum All the cells were incubated at
37C in 5% CO2
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
with primers harboring the mutations, 5¢-CTAATCTCTT CCTCCAAAAAGCACACAGA-3¢ for St-182 and St-93/
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· 104 HEK 293T cells per well in 96-well plates were