The NF-jB p65 and c-Jun activity required for LPS-induced CMV IE enhancer/promoter activation To examine the relative contribution of NF-jB and c-Jun to CMV IE enhancer/promoter activati
Trang 1NF-jB- and c-Jun-dependent regulation of human cytomegalovirus immediate-early gene enhancer/promoter in response to
lipopolysaccharide and bacterial CpG-oligodeoxynucleotides
in macrophage cell line RAW 264.7
Younghee Lee1, Wern-Joo Sohn3, Doo-Sik Kim2,3and Hyung-Joo Kwon3
1 Cell Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Daejon, Korea;
2
Department of Biochemistry and3Institute of Life Science and Biotechnology, College of Science, Yonsei University, Seoul, Korea
The cytomegalovirus immediate-early (CMV IE) gene
enhancer/promoter regulates the expression of
immediate-early gene products and initiation of CMV replication
TNF-a and lipopolysaccharide (LPS) strongly activate the
promoter, possibly involving NF-jB
CpG-oligodeoxy-nucleotides (CpG-ODNs), which contain unmethylated
CpG dinucleotides in the context of particular base
sequences, have gained attention because of their stimulating
effects, via NF-jB, which have a strong innate immune
response To study the effects of LPS and CpG-ODNs, as
well as the mechanisms of their actions regarding CMV IE
enhancer/promoter activation, we used a macrophage cell
line, RAW 264.7 Stimulation of the cells with LPS or
CpG-ODNs resulted in the activation of the CMV IE enhancer/
promoter We examined the involvement of NF-jB and
c-Jun transcription factors by promoter deletion/site-specific
mutation analysis and ectopic expression, and found them to have additive effects Involvement of myeloid differentiation protein, an upstream regulator of NF-jB and c-Jun, was also investigated Experimental results indicate that both LPS-induced and CpG-ODN-induced activations of CMV IE enhancer/promoter are mediated by Toll-like receptor signaling molecules Several lines of evidence sug-gest the potential contribution of bacterial infection in CMV reactivation along with the potential application of CpG-ODNs in gene therapy as a stimulator for the optimal expression of target genes under the control of the CMV IE enhancer/promoter
Keywords: CMV IE gene enhancer/promoter; CpG-ODN; lipopolysaccharide; macrophage; NF-jB
Human cytomegalovirus (CMV) is a ubiquitous
b-herpes-virus that causes severe disease in immunocompromised
patients who have had this virus reactivated from latency
[1] Previous studies suggest that macrophage-lineage cells
may provide a long-lived site for CMV latency [2–4] The
CMV immediate-early (IE) enhancer/promoter regulates
the expression of immediate-early gene products and the
initiation of CMV replication [5,6] The enhancer/promoter
region of the CMV IE gene contains a complexarray of
potential regulatory elements: four types of repetitive
sequence elements of 16, 18, 19 and 21 bp repeats, which
occur three to five times within the enhancer region These
elements contain consensus binding sites for transcription
factors such as NF-jB/Rel, ATF/CREB, YY1 and NF1
[7–13] Furthermore, binding sites for multiple transcription factors, including Sp1 (a serum response element), ELK-1, CCAAT/enhancer binding protein, retinoic acid receptor RAR-RXR family members and AP-1 have been identified [7,12–16]
TNF-a has been identified as a powerful mediator of CMV stimulation and reactivation in human and murine monocyte/macrophage progenitors [3] TNF-a and lipo-polysaccharide (LPS) strongly activate the CMV IE enhancer/promoter, which is mediated by nuclear factor
jB (NF-jB) that binds to the jB sites in the IE enhancer/ promoter [17,18] However, detailed studies are required to understand the mechanisms involved in this action The immediate recognition of bacteria and their pro-ducts in the early line of host defense is mediated by an ancient immune response that uses conserved pattern recognition receptors to distinguish the pathogen-associ-ated molecular pattern signatures of bacterial components [19] A wide variety of bacterial components including LPS, bacterial DNA, peptidoglycan and lipoteichoic acid are capable of stimulating the innate immune responses [20] LPS is the major component of the outer surface of Gram-negative bacteria; it is a typical example of potent activators LPS-induced activation of the innate immune system is mediated through Toll-like receptor (TLR) 4, which is a transmembrane receptor that shares a high degree of homology with TLR9 [21,22] The innate
Correspondence to H.-J Kwon, Institute of Life Science and
Biotechnology, College of Science, Yonsei University,
Seoul 120-749, Korea.
Fax: + 82 2 312 6027, Tel.: + 82 2 2123 6521,
E-mail: hjookwon@yonsei.ac.kr
Abbreviations: CMV IE, cytomegalovirus immediate-early;
CpG-ODNs, oligodeoxynucleotides; IjBaSR, IjBa super repressor;
IL-1R, IL-1 receptor; MyD88, myeloid differentiation protein;
TLR, Toll-like receptor.
(Received 22 November 2003, revised 15 January 2004,
accepted 26 January 2004)
Trang 2immune system recognizes synthetic oligodeoxynucleotides
(CpG-ODNs) and bacterial DNA containing
unmethyl-ated CpG dinucleotides in the context of particular base
sequences (CpG motifs) [23–25] Immune activation by
CpG-ODNs depends on TLR9, which determines the
specificity of CpG motifs [26–28]
Through TLR-dependent signaling pathways in
macro-phages, LPS and CpG-ODNs induce the activation of
several transcription factors including NF-jB [25,29,30]
NF-jB activation requires signal transduction molecules
such as myeloid differentiation protein (MyD88), IL-1
receptor (IL-1R)-associated kinase, tumor necrosis factor
receptor-associated kinase 6 and IjB kinase [30–34]
Sti-mulation of this pathway by LPS and CpG-ODNs results
in the activation of IjB kinase [31,35]; subsequently, IjB
kinase phosphorylates IjBa at serines 32 and 36, leading to
its degradation and the subsequent release and translocation
of NF-jB to the nucleus, where NF-jB facilitates the
transcriptional upregulation of genes downstream from the
jB motif The exposure of macrophages to LPS and
CpG-ODNs also results in the activation of the mitogen-activated
protein kinase pathways, including the extracellular
signal-related kinase, c-Jun NH2-terminal kinase and p38 cascades
[36,37] AP-1 activity is regulated in part by the activation
of c-Jun NH2-terminal kinase which phosphorylates and
subsequently increases the transcriptional activity of c-Jun
in LPS- and CpG-ODN-treated macrophages [37,38] These
common features of the signaling of LPS and CpG-ODNs
suggest that the two stimulators use similar intracellular
pathways to display their shared actions LPS and
CpG-ODNs modulate the expression of many genes that encode
cytokines, cell surface receptors, transcription factors and
proteins related to the proliferation/differentiation of cells
[39,40]
To examine the effects of LPS and CpG-ODNs in cells
that harbor a CMV promoter and to understand the
mechanisms of CMV IE enhancer/promoter activation,
we used a line of RAW 264.7 cells that exhibit
NF-jB-dependent activation of the CMV IE enhancer/promoter
upon LPS or CpG-ODNs stimulation We examined
involvement of NF-jB, c-Jun transcription factors and
MyD88 Our results indicate that LPS-induced and
CpG-ODN-induced signals use TLR/IL-1R signaling molecules
for CMV IE enhancer/promoter activation
Materials and methods
Cell culture and reagents
The RAW 264.7 cell line was obtained from the American
Type Culture Collection (ATCC, Manassas, VA, USA)
Cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% (v/v) fetal bovine serum,
100 UÆmL)1penicillin and 100 lgÆmL)1streptomycin
Viab-ility, which was assayed using trypan blue dye exclusion, was
typically greater than 95% Cultures were maintained until
passage 20 and then discarded Escherischia coli LPS (Sigma,
St Louis, MO, USA) was suspended in sterile water and
added to the cell culture to obtain the desired concentrations
The NF-jB p65 monoclonal antibody was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA)
The rabbit polyclonal antibodies to IjBa and c-Jun were
purchased from Cell Signaling Technology (Beverly, MA, USA) Expression vectors encoding NF-jB p65 (pTL-1 vector), IjBaSR (LxSN vector) and c-Jun (pTL-1 vector) were kindly provided by H Nakshatri (Indiana University, School of Medicine, Indianapolis, IN, USA) An expression vector encoding a dominant negative version of MyD88 (DMyD88) was kindly provided by J Tschopp (University
of Lausanne, Epalinges, Switzerland)
Oligodeoxynucleotides Phosphorothioate backbone oligodeoxynucleotides were purchased from GenoTech (Daejeon, Korea) The CpG-ODN 1826 that we used consisted of 20 bases that con-tained two CpG motifs (underlined): TCCATGCGTT CCTGACGTT Derivatives of the CpG-ODN 1826 se-quence with one or two of the CG sese-quences reversed to GC (indicated by bold lettering) are as follows: CpG-ODN 1826(S-1, TCCATGAGCTTCCTGACGTT); 1826(S-2, TCCATGACGTTCCTGAGCTT) and 1826(S-3, TCC ATGAGCTTCCTGAGCTT) The non-CpG-ODN 2041 (CTGGTCTTTCTGGTTTTTTTCTGG) served as a neg-ative control The LPS content of ODNs was <1 ng LPSÆmg)1DNA as measured by a Limulus amebocyte assay (Whittaker Bioproducts, Walkersville, MD, USA) Construction of luciferase reporter plasmids The CMV IE enhancer/promoter fragments)740 to +65, )507 to +65, )300 to +65, )185 to +65 and )130 to +65 relative to the IE transcription start were amplified by polymerase chain reactions using the entire native CMV enhancer/promoter sequences as a template with the fol-lowing primer sets: 5¢-primers, CMV IE ()740) 5¢-AGGT ACCCAATATTGGCCATTAGCC-3¢; CMV IE ()507) 5¢-CGGTACCTGGCCCGCCTGGCTGAC-3¢; CMV IE
TGGCACCAAAATCA-3¢ and 3¢ primer, CMV IE (+65) 5¢-AAGATCTGACTGCGTTAGCAATTTAAC-3¢ The CMV IE enhancer/promoter fragments were ligated into KpnI and BglII sites of the luciferase reporter plasmid pGL3-basic vector (Promega, Madison, WI, USA) yielding the reporter constructs pCMV-Luc, pCMV()507)-Luc, pCMV()300)-Luc, pCMV()185)-Luc and pCMV()130)-Luc To introduce site-specific mutations in NF-jB and AP-1 binding sites, the transcription factor recognition sites were abrogated and changed to ClaI sites by a two step PCR mutagenesis method [41] using 5¢-primer CMV IE ()740) and 3¢-primer CMV IE (+65) along with the primers encoding the following sequences in sense or antisense orientation: mNF-jB1, 5¢-GTAACGCCAAT AtcGAtTTTCCATTG-3¢; mNF-jB2, 5¢-ACATGACCT TAatcGAtTTTCCTACT-3¢; mNF-jB3, 5¢-GTTTGACT CAatcGatTTTCCAAGTC-3¢; mNF-jB4, 5¢-CCAAAAT CAAatcGatTTTCCAAAATG-3¢; mAP-1, 5¢-TAGCGG TTTatCgatCGGGGATTTCC-3¢ Mutated sites are indi-cated with lower case letters The full-length CMV IE enhancer/promoter sequences ()740 to +65) including the mutations were ligated into the KpnI and BglII sites
of pGL3-basic vector yielding the reporter constructs
Trang 3pCMVmNF-jB1-Luc, pCMVmNF-jB2-Luc, pCMVmN
F-jB3-Luc, pCMVmNF-jB4-Luc, pCMVmAP-1-Luc
Transfection and luciferase assay
One day before the transfection, RAW 264.7 cells were
placed into six-well plates at a concentration of 5· 105cells
per well Cells were transfected using FuGene 6
Transfec-tion Reagent (Roche, Indianapolis, IN, USA) following the
manufacturer’s instructions, in DMEM with 10% (v/v)
FBS For each comparison between constructs, we
con-firmed equivalent transfection efficiency by cotransfecting
the promoterless Renilla luciferase vector pRL-null
(Pro-mega, Madison, WI, USA) as an internal control [42] After
the transfection, the cells were placed in a complete medium
for 24 h prior to LPS (100 ngÆmL)1or as indicated in the
individual experiments) or CpG-ODNs (3 lMor as
indica-ted in the individual experiments) treatment for 6 h or as
indicated in the individual experiments The cells were
harvested, washed and lysed by freeze-thawing three times,
and the luciferase activities were determined using the
Dual-Luciferase Reporter Assay System (Promega, Madison,
WI, USA) with a TD-20/20 luminometer (Turner Designs,
Sunnyvale, CA, USA) according to the manufacturer’s
specifications Individual assays were normalized for Renilla
luciferase activity and the data are presented as the fold
increase in activity relative to empty vector control The
data are from two or three independent experiments
performed in duplicate or triplicate with similar results
Standard errors are indicated
Indirect immunofluorescence assays and confocal
microscopy
We detected NF-jB p65 nuclear localization by indirect
immunofluorescence assays using confocal microscopy as
described previously [43] RAW 264.7 cells (5· 104) were
cultured directly on glass coverslips in 24-well plates After
24 h, the cells were transfected with the IjBa super
repressor (IjBaSR) construct After an additional 24 h,
the cells were fixed with 4% (v/v) paraformaldehyde in
NaCl/Pifor 10 min at room temperature; they were then
permeabilized with 0.2% (v/v) Triton X-100 in NaCl/Pifor
10 min and blocked with a solution of NaCl/Pi, 15% (v/v)
normal donkey serum (Sigma, St Louis, MO, USA) and
0.2% (v/v) Tween-20 Monoclonal antibodies to
NF-jB p65 were applied for 1 h followed by 1 h incubation
with Texas Red-conjugated donkey anti-mouse IgG
(Jack-son ImmunoResearch Laboratories, Inc., West Grove, PA,
USA) For double immunofluorescence staining of
NF-jB p65 and INF-jBa, a primary rabbit polyclonal antibody
that recognizes IjBa was used and detected with a goat
anti-rabbit IgG linked to fluorescein isothiocyanate (Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA,
USA) To identify cell nuclei, we used DNA staining
(0.5 lgÆmL)1of Hoechst no 33258; Sigma, St Louis, MO,
USA) Coverslips were mounted in Fluoromount-G
(South-ern Biotechnology Associates, Inc., Birmingham, AL,
USA) Samples were scanned with a Zeiss LSM 510 laser
scanning confocal device attached to an Axiovert 100
microscope using a Plan-Apochromat 100X/Oil DIC
objective (Carl Zeiss, Germany)
Western blotting Equal amounts of protein were resolved in 10% (v/v) SDS/ PAGE and electrotransferred to poly(vinylidene difluoride) membranes (Millipore, Bedford, MA, USA) Membranes were blocked in NaCl/Tris containing 0.05% (v/v)
Tween-20 and 2% (v/v) BSA for 1 h at room temperature and incubated with appropriate primary antibody for 2 h Immunoreactive proteins were detected by horseradish peroxidase-conjugated secondary antibody (Jackson Immu-noResearch Laborities, Inc.) and an enhanced chemilu-minescence reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA)
Results
Activation of the CMV IE enhancer/promoter
in LPS-treated RAW 264.7 cells
To examine LPS-stimulated CMV promoter activation in RAW 264.7 cells, we used a 740 bp CMV IE enhancer/ promoter-luciferase reporter construct (pCMV-Luc) In the CMV IE enhancer/promoter gene located between the )425 to )80 bp 5¢-region of the transcription start site,
Fig 1 LPS stimulates CMV IE enhancer/promoter in RAW 264.7 cells (A) Structure of the CMV IE enhancer/promoter (B and C) RAW 264.7 cells were transiently transfected with pCMV-Luc for
24 h The cells were then stimulated with increasing amounts of LPS for 6 h (B) or 100 ngÆmL)1LPS for different time periods (C) Cells were harvested and cell lysates were obtained by freeze-thaw The luciferase activity, which was normalized to Renilla activity, was measured as relative light units (RLU) The results are represented as fold activation compared with control vector alone.
Trang 4there are four copies of the NF-jB and one copy of the
AP-1 binding sites (Fig 1A) LPS activates the Toll-like
receptor-dependent signaling pathway in macrophages,
including the transcription factors NF-jB and c-Jun
[32,38] As expected, LPS activated the promoter in a
time-and dose-dependent manner (Fig 1B,C) When the cells
were stimulated with 100 ngÆmL)1 of LPS for 6 h, the
activation of the promoter reached its maximum level
(Fig 1B,C) The following experiments were carried out
under these conditions to estimate the CMV IE enhancer/
promoter activity
The NF-jB p65 and c-Jun activity required for
LPS-induced CMV IE enhancer/promoter activation
To examine the relative contribution of NF-jB and c-Jun to
CMV IE enhancer/promoter activation, we cotransfected
expression plasmids encoding NF-jB p65, c-Jun or both into RAW 264.7 cells with a promoter-reporter construct, and the luciferase activity was estimated First, when the ability of NF-jB p65 to transactivate the reporter was tested, the expressed NF-jB p65 significantly activated the CMV IE enhancer/promoter in a dose-dependent manner (Fig 2A) Cotransfection of an NF-jB p65 expression vector at a concentration of 200 ngÆmL)1conferred about
a five-fold increase in the activation over the empty expression vector control To determine the contribution
of c-Jun to CMV IE enhancer/promoter activation, we introduced the ectopic expression of c-Jun, which also activated the promoter in a dose-dependent manner (Fig 2B) The expression of both NF-jB p65 and c-Jun resulted in an approximately two-fold increase in the stimulation of the transcription compared with the expres-sion of NF-jB p65 or c-Jun alone (Fig 2C) These
Fig 2 Ectopic expression of NF-jB p65 and c-Jun enhances LPS-induced CMV IE enhancer/promoter activation (A and B) RAW cells were transfected with pCMV-Luc and indicated amounts of plasmids expressing NF-jB p65 (A) or c-Jun (B) (C) RAW 264.7 cells were cotransfected with pCMV-Luc, Renilla internal control plasmid and 50 ng of the expression vector encoding either NF-jB p65 or c-Jun or both (D) RAW 264.7 cells were cotransfected with pCMV-Luc and 50 ng of the indicated plasmid constructs Sixhours before harvest, cultures were treated with LPS (100 ngÆmL)1) Luciferase assays were performed as described Experiments represent one of three independent experiments with similar qualitative results (E) Ectopic expression of NF-jB p65 and c-Jun RAW 264.7 cells were transfected with 0.2 lg of NF-jB p65 or c-Jun expression vector After 24 h, cells were stimulated with 100 ngÆmL)1of LPS for 6 h Cell lysates were analyzed by Western blotting with a polyclonal antibody specific for c-Jun and monoclonal antibodies to NF-jB p65 and a-actin.
Trang 5observations indicate that each of the transcription factors
contributes to CMV IE enhancer/promoter activation in
RAW 264.7 cells
Further investigation was performed to examine the
contribution of NF-jB and c-Jun to LPS-dependent
activa-tion of the CMV IE enhancer/promoter Treatment of
RAW 264.7 cells with LPS resulted in an approximately
two-fold increase in the promoter activity The expression of
NF-jB p65 dramatically increased LPS-induced transactivation
of the promoter (Fig 2D) As expected, ectopic expression
of c-Jun also greatly increased the promoter activity in
RAW 264.7 cells treated with LPS (Fig 2D) Taken
together, these data demonstrate that NF-jB p65 and
c-Jun are components of the LPS signaling pathway,
provi-ding further evidence that NF-jB p65 and c-Jun are
essent-ial components of CMV IE enhancer/promoter activation
To exclude the possibility of LPS stimulation affects on
the ectopic expression of NF-jB p65 or c-Jun (pTL-1
vector), we analyzed the lysates from each set of expression
vector-transfected cells following stimulation with LPS for
6 h (Fig 2E) LPS did not change total NF-jB p65 or c-Jun
protein levels We could not detect any dramatic increase in
expression of NF-jB p65 or c-Jun in transfected cells using
Western blotting, probably because of the transfection
efficiency in RAW cells and high expression of endogenous
proteins (Fig 2E) However, we did detect higher expres-sion of NF-jB p65 or c-Jun in a subpopulation of cells transfected with NF-jB p65 or c-Jun expression vector using immunostaining and confocal microscopy images (data not shown) In order to further confirm that pTL-1 vectors do not respond to LPS, we constructed a pTL-1-luciferase reporter (pTL-1-Luc) and examined its regulation
in RAW 264.7 cells after stimulation with LPS LPS did not change the luciferase activity in pTL-1-Luc transfected cells (data not shown)
Inhibition of NF-jB p65 nuclear localization suppresses LPS-induced CMV IE enhancer/promoter activation
To verify whether the CMV IE enhancer/promoter is modulated by LPS dependent nuclear localization of NF-jB p65, it was attempted to analyze the location of NF-jB p65 by immunostaining and confocal microscopy
In RAW 264.7 cells transfected with an empty expression vector and in the absence of exogenous LPS, NF-jB p65 is primarily cytoplasmic as associated with IjBa (Fig 3, first row) The degradation of IjBa and nuclear accumulation
of NF-jB p65 were strongly induced after stimulation with LPS in RAW 264.7 cells transfected with an empty expression vector (Fig 3, second row) In contrast, the
Fig 3 Inhibition of NF-jB p65 nuclear loca-lization by IjBaSR in LPS stimulated RAW 264.7 cells Inhibition of NF-jB p65 nuclear localization by IjBaSR Cells were transfected with the IjBaSR expression vector (0.2 lgÆmL)1) for 24 h and treated with LPS Degradation of IjBa and localization of
NF-jB p65 were visualized by confocal micros-copy after immunofluorescence staining with antibodies to NF-jB p65 (red) and IjBa (green) Cells were stained with Hoechst
no 33258 to visualize the nuclei (blue).
Trang 6expression of a mutant IjBa protein (IjBa super repressor,
IjBaSR) that cannot be phosphorylated on serines 32 and
36 blocked the degradation of IjBa and nuclear
accumu-lation of NF-jB p65 induced by LPS (Fig 3, third row)
To examine the contribution of NF-jB p65 nuclear
localization to LPS-induced CMV promoter activation, an
IjBaSR expressing vector was employed in a
dose-depend-ent manner As expected, the expression of IjBaSR inhibited
activation of the promoter in an IjBaSR dose-dependent
manner (Fig 4A) To confirm the effect of ectopically
expressed IjBaSR on LPS-induced IjBa degradation, the
protein level of IjBa was estimated after the stimulation
Empty expression vector or IjBaSR expression
vector-transfected cell was exposed to LPS for 30 min and then
IjBa was analyzed by Western blot using an antibody against IjBa As shown in Fig 4B, IjBa degradation after LPS treatment was not detected in IjBaSR transfected cells These experimental results indicate that LPS-dependent
NF-jB p65 nuclear localization is critical for activation of the CMV IE enhancer/promoter in RAW 264.7 cells To deter-mine whether LPS stimulation alters ectopic expression of the IjBaSR, Western blotting was performed with the lysates from IjBaSR expression vector-transfected cells after sti-mulation with LPS for 6 h There was no significant variation
in the expression of IjBa in LPS-stimulated cells (Fig 4C) Deletion analysis of the CMV IE enhancer/promoter
To analyze the 5¢-region of the CMV IE enhancer/promo-ter, RAW 264.7 cells were transiently transfected with a series of plasmids containing progressive truncations of the 5¢-promoter sequence between)740 and )130 bp, and luciferase activities were analyzed 6 h later (Fig 5A) The full-length promoter pCMV-Luc had strong luciferase activity, but deletion of 232 bp [)740 to )508 bp, pCMV()507)-Luc] resulted in higher activity than that of pCMV-Luc This finding suggests that negative regulatory elements are located in a region between)740 and )507 bp Deletion up to the )301 bp position did not significantly affect the luciferase activity, whereas progressive deletion
in the region)300 to )130 bp dramatically decreased the activity Therefore, the two NF-jB sites and one AP-1 site located in the region appear to be critical for basal activation of the promoter
To define the LPS-responsive elements of the CMV IE enhancer/promoter, deletion constructs were transiently transfected into RAW 264.7 cells, which were treated with
100 ngÆmL)1 LPS (Fig 5B) Deletion of the upstream sequence of )507 bp resulted in higher LPS-induced promoter activation than that of pCMV-Luc Removal of
a region from)507 to )301 bp [pCMV()300)-Luc], which contains an NF-jB site, reduced LPS-induced promoter activation by 35% In addition, deletion to )185 bp, which lacks another NF-jB site, further reduced the LPS-induced response Furthermore, deletion to )130 bp removed a region that contained the AP-1 and NF-jB sites and abolished the LPS-induced promoter activity These results imply that NF-jB and AP-1 sites are required for the LPS-induced CMV IE enhancer/promoter activity in the macrophage
The contribution of NF-jB and AP-1 binding sites
to the CMV IE enhancer/promoter
To better evaluate the individual contribution of NF-jB and AP-1 to the activity of the CMV promoter, the constructs of the deletion mutant promoter were transiently cotransfected with expression plasmids encoding NF-jB p65 or c-Jun into RAW 264.7 cells (Fig 6) Cotransfection of an NF-jB p65 expression vector at a concentration of 50 ngÆmL)1with a full-length promoter (pCMV-Luc) conferred about three-fold increase in activation over basal transcriptional levels Deletion of the upstream sequence of)507 bp caused the activity of the promoter to be higher than the activity of pCMV-Luc However, progressive deletion of the NF-jB binding sites from the CMV IE enhancer/promoter
Fig 4 Degradation of IjBa required for CMV IE enhancer/promoter
activation in LPS stimulated RAW 264.7 cells (A) Inhibition of
LPS-induced CMV IE enhancer/promoter activation by IjBaSR.
RAW 264.7 cells were cotransfected with pCMV-Luc and indicated
amounts of plasmid expressing IjBaSR The cells were treated with
LPS (100 ngÆmL)1) for 6 h and assayed for luciferase activity (B)
Inhibition of LPS-induced IjBa degradation by IjBaSR Equivalent
amounts of cell extracts from RAW cells transfected with pCMV-Luc
(0.2 lgÆmL)1) and increasing amounts of IjBaSR expression
con-structs were analyzed by Western blotting IjBa and IjBaSR were
detected with the antibody against IjBa (C) Ectopic expression of
IjBaSR in the presence or absence of LPS RAW 264.7 cells were
transfected with 0.2 lg of IjBaSR expression vector After 24 h, cells
were stimulated with 100 ngÆmL)1of LPS for 6 h Cell lysates were
analyzed by Western blotting with polyclonal antibodies specific for
IjBa and a-actin.
Trang 7[pCMV()300)-Luc, pCMV()185)-Luc and
pCMV()130)-Luc)] reduced enhancement of the promoter activity by
NF-jB p65 (Fig 6A) In conclusion, NF-jB and its
recog-nition sites are clearly involved in the CMV IE enhancer/
promoter activation
When the AP-1 site-deleted CMV IE enhancer/promoter
[pCMV()130)-Luc] was cotransfected with a c-Jun
expres-sion vector, the luciferase activities decreased in comparison
with the cells cotransfected with pCMV-Luc (Fig 6B) In
contrast, the deletion of the upstream sequences of)185 bp
[pCMV()507)-Luc, pCMV()300)-Luc and
pCMV()185)-Luc] had no significant effect in c-Jun-induced promoter
activation Cotransfection of both NF-jB p65 and c-Jun
enhanced the activation of the promoters compared with
the activation observed upon transfection of NF-jB p65
or c-Jun alone, in an additive manner (Fig 6C) The
observations indicate that each of the transcription factors
could activate the CMV IE enhancer/promoter in the
mouse macrophage cell line RAW 264.7
We next investigated functional significance of the
tran-scription factors after stimulation with LPS Ectopic
expres-sion of NF-jB p65 or c-Jun significantly increased CMV IE
enhancer/promoter activity in RAW 264.7 cells treated with
LPS (Fig 6D,E) To define the LPS-responsive elements of
the CMV IE enhancer/promoter, the 5¢-deletion series of constructs were transiently cotransfected with expression plasmids encoding NF-jB p65 or c-Jun into RAW 264.7 cells, which were then treated with 100 ngÆmL)1LPS The expression of NF-jB p65 or c-Jun dramatically increased the LPS-induced transactivation in the two promoter-reporter constructs pCMV-Luc and pCMV()507)-Luc (Fig 6D,E) However, this phenomenon was greatly reduced when the promoter sequence upstream of )300 bp was deleted [pCMV()300)-Luc] Progressive deletion of the region including NF-jB and AP-1 binding sites [pCMV()300)-Luc, pCMV()185)-Luc and pCMV()130)-Luc] reduced the promoter activity stimulated by LPS in the presence of ectopically expressed NF-jB p65 or c-Jun (Fig 6D,E) This result indicates that the region, between)507 and )130 bp of the CMV IE enhancer/promoter including NF-jB and AP-1 binding sites, is required for NF-jB p65 or c-Jun to enhance the LPS-induced transcriptional activation
Effects of site-specific mutations in the NF-jB or AP-1 binding sites on the CMV IE enhancer/promoter activity
To further clarify the individual roles of NF-jB and AP-1 sites on activation of the CMV IE enhancer/promoter, we
Fig 5 Effects of the 5¢-promoter sequence deletion mutations on the activity of the CMV IE enhancer/promoter (A) Relative activities of the CMV IE enhancer/promoter constructs RAW cells were transfected with each reporter construct and cultured for 24 h before assaying for luciferase activity The results are presented as relative activation compared with the pCMV-Luc construct (B) LPS induction of the promoter constructs RAW cells were transfected with each reporter construct and cultured in the presence or absence of LPS (100 ngÆmL)1) for 6 h The fold activation represents the ratio of luciferase activity in LPS stimulated cells versus unstimulated cells Essential regions for basal promoter activity (A) and LPS-responsiveness (B) were indicated.
Trang 8performed mutational analysis of these sites in the context
of the full-length promoter (Fig 7) The full-length
promo-ter pCMV-Luc had strong luciferase activity and mutation
of the NF-jB1 site had minimal effect on the promoter
activity Mutation in the NF-jB2, NF-jB3 and NF-jB4
sites reduced the promoter activity by 62, 72 and 51%,
respectively (Fig 7B) Mutation in the AP-1 binding site
also reduced promoter activity by 27% (Fig 7B)
There-fore, the three NF-jB sites and one AP-1 site appear to be
critical for basal activation of the promoter
To determine which transcription factor binding sites are
required for maximal induction of the CMV IE enhancer/
promoter by LPS, mutant constructs were transiently
transfected into RAW 264.7 cells, which were treated with
100 ngÆmL)1 LPS Mutation in the NF-jB1 site had
minimal effect on the LPS-induced promoter activity The
NF-jB1 site is located in the region between )507 and
)300 bp of the CMV IE enhancer/promoter which, based
on promoter deletion analysis, is required for the
LPS-induced transcriptional activation (Figs 5B and 6D,E)
Therefore, it is likely that factor(s) other than NF-jB and
AP-1 may be also involved in LPS-induced promoter
activation Significant reduction of the promoter activity
was observed by mutation of NF-jB2, NF-jB3, NF-jB4
and AP-1 sites in LPS-stimulated cells (Fig 7C) We
conclude that LPS activation of CMV IE
enhancer/promo-ter is mediated by a concerted action of transcription factor
binding sites and each of the NF-jB and AP-1 sites is
important for maximal activation
We investigated involvement of NF-jB p65 and c-Jun in
transcriptional activation of the CMV IE
enhancer/promo-ter using the site-specific mutant constructs (Fig 7A) and
ectopic expression of NF-jB p65 or c-Jun (Fig 7D,E)
Mutations in the NF-kB binding sites from the CMV IE
enhancer/promoter (jB2-Luc,
pCMVmNF-jB3-Luc and pCMVmNF-jB4-Luc) reduced promoter
activation after ectopic expression of NF-jB p65 (Fig 7D)
Among the four NF-jB binding sites, NF-jB3 appears
to have the most significant effect on the transcriptional
activation When AP-1 site was mutated, promoter activity
after ectopic expression of c-Jun was decreased compared to
wild type promoter Taken together, these results confirm
that the NF-jB and AP-1 sites both contribute to CMV IE
enhancer/promoter activity
Activation of the CMV IE enhancer/promoter
by CpG-ODN
To evaluate the effects of CpG-ODNs on CMV IE
enhancer/promoter activation, we used a CMV IE
enhan-cer/promoter-luciferase reporter (pCMV-Luc) and ex am-ined its regulation in RAW 264.7 cells after stimulation with CpG-ODN 1826, which consists of 20 bases containing two CpG motifs The CpG-ODN 1826 activated the CMV IE
Fig 6 Activation of the CMV IE enhancer/promoter constructs in
response to ectopic expression of NF-jB p65 and c-Jun (A, B and C)
RAW cells were transfected with each reporter construct (0.2 lg each)
and the indicated expression vectors (50 ng each) encoding NF-jB p65
(A), c-Jun (B) or both (C) and cells were cultured for 24 h before
assaying for luciferase activity (D and E) RAW 264.7 cells were
cotransfected with each reporter construct and 50 ng of the indicated
expression vectors Six hours before harvest, cultures were treated with
LPS The results are presented as fold activation compared with the
control expression vector.
Trang 9enhancer/promoter in a time- and dose-dependent manner
(Fig 8A,B) Activation of the promoter reached its
maxi-mum level when stimulated with 3 lMof the CpG-ODN for
6 h (Fig 8A,B) Because CpG-ODN 1826 has two CpG
motifs, we decided to identify the contribution of the
individual CpG dinucleotide sequence to the promoter
activation We synthesized the phosphorothioate backbone
oligonucleotides of CpG-ODN 1826(S-1), 1826(S-2) and
1826(S-3), which differ only in the reversal of the CpG
dinucleotides to the GpC dinucleotides compared to the
CpG-ODN 1826(S) sequence As shown in Fig 8C,
luci-ferase activity decreased by 15% when the cells were
treated with CpG-ODN 1826(S-2) compared to the activity
in ODN 1826(S)-treated cells Compared with
CpG-ODN 1826(S), a much lower level of luciferase activity (up
to 40% reduction) was induced by CpG-ODN 1826(S-1)
With the reversal of the two CpG dinucleotides to GpC
dinucleotides, CpG-ODN 1826(S-3) lost its capacity for
CMV promoter activation Basal luciferase activity was
detected in the control cells stimulated with
non-ODN 2041 These results clearly indicate that the
CpG-ODN 1826 sequence induced CMV IE enhancer/promoter
activation in a CpG sequence-dependent manner
Regulation of CMV IE enhancer/promoter activity
by CpG-ODN and LPS via MyD88-dependent modulation
of NF-jB activation The signaling by CpG-DNA and LPS through their respective TLRs requires the participation of the adaptor protein MyD88 and results in activation of the common transcription factors NF-jB and AP-1 [32,34] To determine
if MyD88 is involved in the CMV IE enhancer/promoter activation, we cotransfected an expression plasmid encoding
a mutant MyD88 (DMyD88) into RAW 264.7 cells, along with the promoter Acting as a dominant negative molecule
in TLR/IL-1R-dependent signaling, the mutant inhibited LPS-mediated and CpG-ODN1826-mediated CMV IE enhancer/promoter activation (Fig 9A,B) We then exam-ined whether the promoter activation is modulated by CpG-ODN signaling-dependent IjBa degradation When RAW 264.7 cells were transfected with a mutant IjBa protein (IjBaSR), activation of the promoter by CpG-ODN 1826 stimulation was inhibited, further confirming that the degradation of IjBa is necessary for CMV IE enhancer/ promoter activation that is induced by CpG-ODN 1826 (Fig 9B)
Fig 7 Effects of site-specific mutations in transcription factor recognition site on the activity of the CMV IE enhancer/promoter (A) Positions and sequences of the NF-jB and AP-1 sites are indicated Mutated sites are indicated with lower case letters (B) Relative activities of the mutated CMV IE enhancer/promoter construct RAW cells were transfected with each mutated reporter construct and cultured for 24 h before assaying for luciferase activity The results are presented as relative activation compared with the pCMV-Luc construct (C) LPS induction of the promoter constructs RAW cells were transfected with each mutated reporter construct and cultured in the presence or absence of LPS (100 ngÆmL)1) for 6 h, (D and E) RAW cells were transfected with each mutated reporter construct (0.2 lg each) and the indicated expression vectors (0.2 lg each) encoding NF-jB p65 (D), c-Jun (E) and cultured for 24 h before luciferase activity assay The results are presented as relative activation compared with the pCMV-Luc construct.
Trang 10Although many investigators use vectors such as pRC/CMV
and pcDNA to introduce target genes under the control of a
CMV promoter, only a few studies have revealed regulation
of the promoter in detail Reportedly, interferon-a inhibits
murine CMV immediate-early gene expression by
downreg-ulating NF-jB activity [44] In contrast, TNF-a and LPS
have been shown to stimulate CMV IE enhancer/promoter
activity by upregulating NF-jB activity [17,18] In this study,
we demonstrate that LPS and CpG-ODNs activate the
CMV IE enhancer/promoter and that both NF-jB and
c-Jun contribute to this phenomenon in the mouse
macro-phage cell line RAW 264.7
LPS stimulation drastically induced activation of
the CMV IE enhancer/promoter (Fig 1) In other studies,
the NF-jB and c-Jun binding sites were identified in the
CMV IE enhancer/promoter [7,10,12] Our study extends this observation by identifying the dominant role of ectopically expressed NF-jB p65 The expression of NF-jB p65 in RAW 264.7 cells led to an increase of the CMV IE enhancer/promoter basal activity in a dose-dependent fashion (Fig 2A), while NF-jB p65 enhanced the capacity of LPS to dramatically induce the promoter activation (Fig 2D) Using reporter gene assays, we directly demonstrated the major role of NF-jB; preventing nuclear localization of NF-jB by ectopic expression of its specific inhibitor IjBaSR blocked activation of the CMV IE enhancer/promoter in an IjBaSR dose-dependent manner (Figs 3 and 4A) These results suggest that NF-jB activa-tion is required for CMV IE enhancer/promoter activaactiva-tion
in the LPS-signaling pathways Additionally, we confirmed the involvement of NF-jB by using the deletions and site-specific mutations of the promoter region to interfere with NF-jB-mediated activation at the level of DNA binding (Figs 5–7), which suggests that NF-jB and its binding sites are essential for NF-jB-dependent CMV IE enhancer/ promoter activation
The potential function of c-Jun in regulating the CMV IE enhancer/promoter is not fully characterized Accordingly,
Fig 8 CpG-ODN 1826 stimulates activation of the CMV IE enhancer/
promoter in RAW 264.7 cells RAW 264.7 cells were transiently
trans-fected with CMV IE enhancer/promoter-luciferase construct
(pCMV-Luc) for 24 h The cells were then stimulated with increasing amounts of
CpG-ODN 1826 for 6 h (A) or CpG-ODN 1826 (3 l M ) for different
time periods (B) RAW 264.7 cells were transfected with pCMV-Luc
and treated with 3 l M of the indicated CpG-ODNs for 6 h and
luciferase assays were performed (C).
Fig 9 LPS-induced and CpG-ODN 1826-induced CMV IE enhancer/ promoter activity in RAW 264.7 cells is inhibited by IjBaSR and the dominant negative mutant of MyD88 RAW 264.7 cells were transiently cotransfected with the CMV IE enhancer/promoter-luciferase con-struct (pCMV-Luc) and LxSN control vector, IjBaSR or the dom-inant negative mutant of MyD88 for 24 h The cells were treated with
100 ngÆmL)1LPS (A) or 3 l M of CpG-ODN 1826 (B) for 6 h before assaying for luciferase activity The results are represented as fold activation compared with the control vector alone.