From the Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Edobashi, Tsu, Mie 514-8507, Japan
Received for publication, November 14, 2000, and in revised form, March 20, 2001
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ABSTRACT |
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An increase in the intracellular
Ca2+ concentration controls a diverse range of cell
functions, including gene expression, apoptosis, adhesion, motility,
and proliferation. We have investigated Ca2+ regulation of
gene expression in rat aortic smooth muscle cells. We found that the
expression of nuclear factor regulated by interleukin 3 (NFIL3)/adenovirus E4 promoter-binding protein (E4BP4)/basic region/leucine zipper (bZIP) type of a transcription factor that has a
very important function in cell survival, was activated by thapsigargin
(TG). This activation was inhibited by chelation of extra- or
intracellular Ca2+, suggesting that the induction by TG was
dependent on the elevation of [Ca2+]i. Specific
inhibition of calcineurin or calcium/calmodulin-dependent protein
kinase (CaM kinase) by chemical means impaired the TG-induced NFIL3/E4BP4 expression. Expression of dominant negative forms of
calcineurin or nuclear factor of activated T cells (NFAT)
inhibited the induction of NFIL3/E4BP4 mRNA by TG. These results
suggest that intracellular Ca2+ plays a critical role in
regulating gene expression of NFIL3/E4BP4 by calcineurin/NFAT and CaM
kinase signaling in vascular smooth muscle cells.
Calcium signals regulate numerous cell functions, including gene
expression, some forms of programmed cell death, motility, secretion,
proliferation, and cell survival (1).
([Ca2+]i can be
raised experimentally by a number of agents, including the microsomal
calcium ATPase inhibitor TG1 (1, 2). We investigated the
regulation of gene expression in rat aortic smooth muscle cells (RASMC)
exposed to TG by utilizing the fluorescent differential display
technique (3). We compared the complementary DNA fragments from
differentially expressed RASMC mRNAs following 3 h treatment
with either vehicle or 100 nM TG. This analysis resulted in
the identification of the rat homologue of NFIL3/E4BP4 as a
TG-inducible gene.
NFIL3/E4BP4 was originally isolated as a novel member of the bZIP
family of DNA-binding proteins that displays an unusual DNA binding
specificity which overlaps that of the activating transcription
factor family and cAMP response element-binding protein (4).
NFIL3/E4BP4 repressed promoter activity and this repression was
mediated through the cAMP response element/activating transcription
factor-like site (5). NFIL3/E4BP4 might play a role in the
glucocorticoid repression of several genes, because NFIL3/E4BP4 was
induced by the synthetic glucocorticoid dexamethasone (6). Moreover,
NFIL3/E4BP4 repressed the stimulating activity of box NFIL3/E4BP4 also plays an important role in the expression of IL-3 in T
cells (8). In mouse pro-B cell lines, NFIL3/E4BP4 was also regulated as
a delayed-early IL-3 responsive gene, requiring de novo
protein synthesis (9). In the absence of IL-3, enforced expression of
human NFIL3/E4BP4 promoted the survival but not the growth of
IL-3-dependent pro-B cells, indicating that induction of
NFIL3/E4BP4 is one of the mechanisms through which IL-3 suppresses apoptosis (9).
Recently, it has been found that the expression of NFIL3/E4BP4 was
regulated by oncogenic Ras mutant proteins through both the
Raf-mitogen-activated protein kinase and the phosphatidylinositol 3-kinase pathways in murine Pro-B lymphocytes (10). Ras-NFIL3/E4BP4 pathways may be common targets for a variety of oncogenes (10).
In this paper, we describe the cloning and calcium regulation of
expression of rat NFIL3/E4BP4 in RASMC. We show that TG induced rat
NFIL3/E4BP4 gene expression by activating the
calcineurin/NFAT and by CaM kinase signaling. Thus, calcium signaling
may be critical for the regulation of NFIL3/E4BP4 through
calcineurin/NFAT and CaM kinase signaling in vascular smooth muscle cells.
Reagents--
Thapsigargin, calcium ionophore A23187, phorbol
12-myristate 13-acetate (PMA), actinomycin D, and cycloheximide were
purchased from Sigma. Cyclosporin A, FK506, KN-93, and PD98059 were
purchased from CalBiochem. KN-92 was purchased from SeikagakuKogyo Co.
Tokyo, Japan.
Cloning of Rat NFIL3/E4BP4 cDNA--
For cloning the
3'-half of the rat NFIL3/E4BP4 cDNA, we performed a rapid
amplification of cDNA ends (RACE) (11). The 3'-half of the
full-length cDNA was obtained using the primers
5'-TTACCGCACAAGCTCCGGATTAAAG-3' and 5'-CCATCCTAATACGACTCACTATAGGGC-3'
for the first RACE and 5'-GCAGATGCGCTAGCCAAAAGACATT-3' and
5'-ACTCACTATAGGGCTCGAGCGGC-3' for the second RACE. A rat heart Marathon
cDNA amplification kit (CLONTECH) was used for
the template. Unfortunately, we could not obtain the 5'-half of the
full-length cDNA by RACE, and so we performed polymerase chain
reactions (PCR) using a degenerate primer. The 5'-half of the
full-length cDNA was obtained by using the primers
5'-AAGGMKYCTGACRGATTTAYCC-3' and 5'-ATGTTCGTCACCTGCACCGAGAAAG-3'. The
PCR products were sequenced using an automated DNA sequencer (Applied Biosystems).
Northern Blotting Analysis--
Northern blotting analysis was
carried out using Rat Multiple Tissue Northern blots
(CLONTECH). 32P-Random prime-labeled
fragment of the rat NFIL3/E4BP4 cDNA (nucleotides 1093-1701) probe
was synthesized with Strip-EZ DNA (Ambion). The heat-denatured cDNA
probe was added at 2 × 106 cpm/ml to ExpressHyb
hybridization solution (CLONTECH). The probe was
hybridized to the RNA blots for 1 h at 65 °C. The membrane was
washed according to the manufacturer's protocol and exposed to x-ray
film at Plasmid Constructs--
The dominant negative human calcineurin
(12) and NFAT (13) were constructed as described previously. Briefly,
the human calcineurin A2 coding region (aa 1-407) and
human NFAT3 coding region (aa 1-160) were obtained by PCR using a
human heart Marathon cDNA amplification kit
(CLONTECH) as template. The PCR products of
calcineurin A2 and NFAT3 were subcloned into the
EcoRI and NotI sites of the plasmid vector
pcDNA.3.1/His (Invitrogen). The pcDNA-Luc expression vector was
prepared by subcloning the luciferase coding region into the
EcoRI and NotI sites of pcDNA.3.1/His
expression vector. The sequence of all PCR-derived constructs was
confirmed by automated DNA sequencing.
Site-directed Mutagenesis--
In vitro
directed mutagenesis was performed by using the commonly used
overlap-extension method (14). Synthetic oligonucleotide primers
containing the desired mutations and complementary to opposite strands
of the corresponding plasmid constructs were extended by using KOD-plus
DNA polymerase (TOYOBO), generating mutated plasmids. Primers used were
pcD-CNA2 (H101Q) mutated primer: 5'-TGTGTGGTGACATCCAgGGCCAATTTTTTG-3',
pcD-CNA2 (H290Q) mutated primer: 5'-TTATTAGAGCTCAgGAAGCTCAAGATGCAG-3',
pcD-NFAT3 (AxIxIA) mutated primer: 5'-
GAGTGTgCCAGCATCCGCATCgCCTCCATC-3', and pcD-NFAT3 (AxAxIA) mutated
primer: 5'- GAGTGTgCCAGCgcCCGCATCgCCTCCATC-3'. Lowercase letters in the
sequence of primers indicated mutated positions. Mutagenesis of more
than one site was done by subsequent mutation of the corresponding
previously mutated construct. The nucleotide sequence of the constructs
was confirmed by automatic DNA sequencing.
Cell Culture and DNA Transfection--
Rat aortic smooth muscle
cells (A7r5 cells, CRL-1444) were obtained from the American Type and
Culture Collection. A7r5 cells were grown in Dulbecco's modified
Eagle's medium (Life Technologies) supplemented with 10%
heat-inactivated fetal calf serum (Life Technologies), 25 µg/ml
streptomycin, and 25 units/ml penicillin (Life Technologies). Cultures
were maintained at 37 °C in a humidified atmosphere of 95% air, 5%
CO2. All experiments were conducted on cells at passages
2-5. Quiescent A7r5 cells were obtained by incubation of preconfluent
cell cultures in Dulbecco's modified Eagle's medium supplemented with
0.5% fetal calf serum for 48 h after which drugs were added to
the culture medium.
For transient transfection experiments, A7r5 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum in a 25-cm2 flask. Cells in the logarithmic growth
phase were transfected in 2 ml of Dulbecco's modified Eagle's medium
plus 0.5% fetal calf serum using 1 µg of the indicated plasmid, 1 µg of pcDNA-Luc, and 8 µl of Tfx-50 (Promega). Transfected A7r5
cells were incubated for 48 h and exposed to vehicle or 30 nM TG for 3 h.
RNA Isolation and cDNA Synthesis--
Total RNAs were
isolated and purified by using ISOGEN (Nippon Gene) reagent according
to the manufacturer's instructions. Total RNAs (0.5-1 µg) were
converted to cDNA using oligo(dT)11-18 primer
(Amersham Pharmacia Biotech) and Superscript II reverse transcriptase
(Life Technologies) according to the manufacturer's protocol. After
synthesis of cDNA, the reaction mixtures were diluted 5-fold by
addition of TE (10 mM Tris, 1 mM EDTA, pH 8.0) for real time RT-PCR.
Real Time RT-PCR--
To monitor mRNA expression, we used
real time RT-PCR analysis. This novel approach has been described
previously (15, 16). For rat NFIL3 real time RT-PCR analysis, the
following primers were used; rNFIL3U, 5'-AAGGGCCCCATCCATTCT-3'; and
rNFIL3L, 5'-TGAGGGACCAATCTTGAA-3'. The fluorescent probe for the real
time RT-PCR was rNFIL3P, 5'-AAAGCCAAGGCCATGCAGGTCAA-3'. The GAPDH
primers and probe (TaqMan GAPDH detection reagents) were purchased from
Applied Biosystems. For luciferase real time RT-PCR analysis, the
following primers were used: LucU, 5'- TGTCGCTCTGCCTCATAGAACTG-3'; and
LucL, 5'- CAGCAGCGCACTTTGAATCTTG-3'. The fluorescent probe for the real
time PCR was LucP: 5'- CATTCCGGATACTGCGATTTTAAGTGT-3'. The RT-PCR
reaction and the resulting relative increase in reporter fluorescent
dye emission were monitored in real time by the 7700 sequence detection
program (Applied Biosystems). Conditions were as follows: 1 cycle at
50 °C for 10 min, 1 cycle at 94 °C for 15 min, 40 cycles at
94 °C for 30 s, 60 °C for 1 min.
Cloning of Rat NFIL3/E4BP4--
To study the effect of elevated
intracellular Ca2+ concentration on gene expression, RASMC
were treated with 100 nM TG for 3 h. RNA was
isolated from non-stimulated or TG-stimulated RASMC and subjected to a
fluorescent differential display (3). A highly induced band was
reproducibly displayed when the primer pair G(T)15A and
5'-GAAACGGGTG-3' was used. This band was cut from the gel,
re-amplified, and subjected to DNA sequence analysis. Comparison the
nucleotide sequence with GenBankTM/EMBL/DDBJ DNA data bases
revealed 74% identity with human NFIL3/E4BP4 (4, 8) and 94% identity
with mouse NFIL3/E4BP4 (9). These results suggest that the clone might
be a rat homologue of NFIL3/E4BP4. To obtain the full-length cDNA
of rat NFIL3/E4BP4, we used polymerase chain reaction. The 3'-half of
the full-length cDNA was obtained by the 3'-RACE technique. Because
we could not obtain any of the 5'-half of the cDNA using RACE, we
designed a degenerate primer from the mouse NFIL3/E4BP4 sequence (clone
IMAGE: 1331717) to clone the 5'-half. DNA sequencing of several
subcloned PCR-generated cDNAs revealed a long open reading frame
commencing with an ATG codon 127 base pairs from the 5' end of the
cDNA sequence (Fig. 1). This ATG
presumably represents the initiation codon, since a TGA stop codon is
positioned 69 base pairs upstream of this triplet in the cDNA
sequence. The open reading frame terminated at a TAA stop codon and was
followed by 315 base pairs of 3'-untranslated region, including a
canonical AATAAA polyadenylation signal. The molecular mass of the rat
NFIL3/E4BP4 was calculated to be 50.8 kDa from an open reading frame
containing 462 amino acids.
Comparison of the deduced amino acid sequences of rat, mouse (9), and
human NFIL3/E4BP4 (4, 8) (Fig. 2)
revealed that NFIL3/E4BP4 is highly conserved among these species (83% identity between rat and human and 95% identity between rat and mouse). The characteristic features of this family are a region of
basic amino acids that could bind DNA (aa 79-93) (17), a region
containing leucine residues repeated every seven amino acids (aa
101-122) (18), and a novel transcriptional repressor domain (aa
299-363) (19). All these characteristic regions are well conserved
across rat, mouse, and human NFIL3/E4BP4.
Tissue Distribution of Rat NFIL3/E4BP4 mRNA--
Northern
hybridization was performed to analyze the tissue distribution of rat
NFIL3/E4BP4 (Fig. 3). A 2.5-kilobase
transcript could be detected in all tissues except the pancreas. Modest
expression was seen in heart, lung, and skeletal muscle. Strongest
expression was seen in liver, although human E4BP4 is expressed very
little in the liver (7). The blot was subsequently reprobed with a radiolabeled rat Induction of NFIL3/E4BP4 mRNA by TG Treatment--
Real-time
RT-PCR analysis was then performed to confirm the differential display
results indicating that rat NFIL3/E4BP4 was a TG-inducible gene. As
shown in Fig. 4A, treatment
with TG resulted in a marked increase in rat NFIL3/E4BP4 mRNA
levels. An elevation in NFIL3/E4BP4 mRNA levels was apparent within
3 h, peaked at 6 h of treatment (20-fold) and expression
returned to almost basal levels by 12 h.
Dose dependence of NFIL3/E4BP4 mRNA induction by TG was also
examined. The induction was increased from 1 to 30 nM with
almost no further increases at higher concentrations (Fig.
4B).
TG-induced Expression of NFIL3/E4BP4 mRNA Is Dependent on
Elevation of Intracellular Ca2+--
TG induces both
elevations of intracellular Ca2+ concentration (2) and an
endoplasmic reticulum (ER) stress response (20). We therefore tested
whether a range of different stress-inducing conditions induced
NFIL3/E4BP4. RASMC were exposed to one or the other of the following
for a period of 3 h: 300 nM TG, 100 nM calcium ionophore A23187, or 5 µg/ml tunicamycin, an inhibitor of
N-linked glycosylation. After treatment, RNA was isolated
from drug-treated cells and real time RT-PCR analysis was performed to
measure rat NFIL3/E4BP4 mRNA levels. NFIL3/E4BP4 mRNA was
induced by TG (12-fold) and A23187 (10-fold). Tunicamycin also induced the expression of NFIL3/E4BP4 mRNA (2.8-fold), but the induction was much less than by TG and A23187 (Fig.
5A). Both TG and A23187
deplete the ER of stored Ca2+ and cause an influx of
Ca2+ into the cells leading to a sustained rise in
cytosolic Ca2+ concentration (1). The depletion of stored
Ca2+ in ER by TG and A23187 also induced the ER stress
response (20, 21). On the other hand, tunicamycin inhibits correct folding and assembly of glycoproteins within the ER and thus also activates the ER stress responses (20). These results suggest that
induction of NFIL3/E4BP4 may occur predominantly as a result of
elevation of the cytosolic Ca2+ concentration.
To further support a role for elevation of cytosolic Ca2+
concentration mediating gene induction by TG, we next examined whether blocking the increase in cytosolic Ca2+ concentration could
prevent expression of the NFIL3/E4BP4 mRNA. As shown in Fig.
5B, pretreatment of A7r5 cells with the extracellular Ca2+ chelating agent EGTA resulted in significant
inhibition of TG-induced expression of NFIL3/E4BP4. Almost complete
inhibition was observed when A7r5 cells were treated with 10 µM BAPTA-AM, a cell permeable Ca2+ chelating
agent (Fig. 5B). These results suggest that increased cytosolic Ca2+ concentration is necessary to induce
NFIL3/E4BP4 gene expression by TG.
Effects of inhibitors of
Ca2+/Calmodulin-dependent Protein Phosphatase
and Protein Kinases on the Induction of NFIL3/E4BP4 mRNA by
TG--
Because increased cytosolic Ca2+ concentration is
necessary to induce NFIL3/E4BP4 gene expression by TG, we
investigated the effects of
Ca2+/calmodulin-dependent protein phosphatase
and protein kinases on NFIL3/E4BP4 expression. Cyclosporin A (CsA) and
FK506, in complex with their immunophilin receptors, specifically
inhibit the Ca2+/CaM-dependent protein
phosphatase, PP2B, also known as calcineurin (22). These two inhibitors
were investigated along with KN-93, an inhibitor of CaM kinase II (23)
(Fig. 6). In the absence of TG, CsA,
FK506, or KN-93 did not significantly influence NFIL3/E4BP4 mRNA
levels. In the presence of TG, both CsA and FK506 significantly inhibited the increase in NFIL3/E4BP4. Moreover, KN-93 markedly reduced
the increase in NFIL3/E4BP4 mRNA by TG. In contrast, KN-92, an
inactive analog of KN-93, displayed much less inhibition than KN-93.
These results suggest that the calcium dependent activation of
NFIL3/E4BP4 gene expression might be mediated by calcineurin and CaM kinase.
TG-induced Expression of NFIL3/E4BP4 Is Transcriptional--
Next,
we explored the effect of the RNA synthesis inhibitor actinomycin D on
the induction of NFIL3/E4BP4 mRNA levels by TG. A7r5 cells were
treated with TG for 3 h, with or without pretreatment with
actinomycin D. RNA was isolated, and NFIL3/E4BP4 mRNA levels were
analyzed by real-time RT-PCR. Pretreatment with actinomycin D
completely prevented the induction of NFIL3/E4BP4 mRNA by TG (Fig.
7). Thus, the increase in NFIL3/E4BP4
mRNA expression after TG addition is due, at least in part, to
transcriptional activation of the NFIL3/E4BP4 gene.
We next used the protein synthesis inhibitor cycloheximide to determine
whether TG-induced expression of NFIL3/E4BP4 mRNA was dependent on
de novo protein synthesis. As shown in Fig. 7, A7r5 cells
were either left untreated or treated with TG for 3 h with or
without pretreatment of cycloheximide. The pretreatment of
cycloheximide to TG-stimulated cells did not significantly prevent
NFIL3/E4BP4 gene induction (Fig. 7). These results indicate that the induction of NFIL3/E4BP4 mRNA by TG does not require de novo protein synthesis for transcriptional activation.
TG Regulates NFIL3/E4BP4 Gene Expression by Mechanisms Involving
Calcineurin--
The immunosuppressive drugs CsA and FK506 target the
phosphatase activity of calcineurin. But it has also been reported that CsA and FK506 could target other proteins (e.g. JNK (24) and p38(25)). To further verify that calcineurin is involved in TG-induced expression of NFIL3/E4BP4 mRNA, we transfected dominant negative calcineurin (12) into A7r5 cells. Calcineurin dephosphorylates NFAT
proteins and induces their translocation from the cytoplasm into the
nucleus. The dominant negative calcineurin is exclusively cytoplasmic
and interferes with NFAT translocation (12). As shown in Fig.
8, the dominant negative calcineurin
(H101Q/H290Q) caused inhibition of TG-induced expression of
NFIL3/E4BP4 mRNA compared with the control. These data indicate
that calcineurin activity is required for the induction of NFIL3/E4BP4
mRNA expression by TG.
TG Regulates NFIL3/E4BP4 Gene Expression by Mechanisms Involving
NFAT--
Next, the involvement of NFAT in the regulation of
NFIL3/E4BP4 expression by TG was analyzed by using dominant negative
NFAT (dnNFAT) (13). The dnNFAT selectively inhibited NFAT transcription activity by interfering with the activation-induced nuclear import of
NFAT through the consensus sequence PxIxIT (13, 26). Interfering with
the docking of calcineurin at the PxIxIT sequence impairs NFAT
activation and NFAT-dependent reporter gene expression (13, 27). As shown in Fig. 9, the induction of
NFIL3/E4BP4 mRNA by TG was markedly reduced in cells transfected
dnNFAT (PxIxIT) although significantly less so with the mutated
construct (AxIxIA). This inhibitory effect was similar to the case of
the inhibition of IL-2 production by the dnNFAT (13). These data
indicate that NFAT transcription activity is required for
NFIL3/E4BP4 gene expression induced by TG.
This study has demonstrated that the expression of NFIL3/E4BP4
mRNA is induced by calcium signaling pathways.
The endoplasmic reticulum calcium-ATPase inhibitor TG depletes
Ca2+ from internal stores, leading to Ca2+
influx and thus, to a sustained increase in
[Ca2+]i (1). TG also induces an ER stress
response (20). We demonstrated that chelation of extracellular and
intracellular Ca2+ by EGTA and BAPTA-AM, respectively,
suppressed the induction of NFIL3/E4BP4 mRNA by TG. We also
demonstrated that the induction of NFIL3/E4BP4 mRNA by tunicamycin,
which induces an ER stress response (20) without disturbing
Ca2+ homeostasis, was less than with TG or A23187. These
results suggest that an increase in [Ca2+]i is
critically involved in NFIL3/E4BP4 mRNA induction by TG.
Fluxes in the amount of intracellular Ca2+ are important
determinants of gene expression (1). Of the many types of
Ca2+-binding proteins, calmodulin (CaM) is of major
importance in vascular smooth muscle cell function.
Ca2+/CaM regulates functional proteins such as calcineurin
(28) and CaM kinase (29) that play important roles in gene expression in vascular smooth muscle cells. To evaluate the relative regulatory roles of calcineurin and CaM kinase, we tested the effect of inhibitors of calcineurin and CaM kinase on TG-induced NFIL3/E4BP4 mRNA
expression. We have demonstrated that both CsA and FK506 inhibited the
induction of NFIL3/E4BP4 mRNA. The induction of NFIL3/E4BP4
mRNA by TG was also inhibited by KN-93. These results indicate that
TG induces the NFIL3/E4BP4 gene expression by calcineurin
and CaM kinase-dependent mechanisms.
To further verify that TG involves calcineurin in the induction of
NFIL3/E4BP4 mRNA, we transfected dominant negative calcineurin into
A7r5 cells. Expression of dominant negative calcineurin strongly suppressed the induction of NFIL3/E4BP4 mRNA by TG. All of these results strongly suggest that calcineurin plays a critical role in the
selective induction of NFIL3/E4BP4 mRNA by TG. Calcineurin regulates immune response genes through dephosphorylation of NFAT (28).
Following dephosphorylation, NFAT is translocated into the nucleus
where it binds either directly to DNA or in a complex with members of
the bZIP transcription factors family such as AP-1 (30) and
subsequently activates gene transcription. NFAT proteins are present in
diverse cell types including vascular smooth muscle cells (31).
We tested the involvement of NFAT in NFIL3/E4BP4 mRNA expression by
using the dnNFAT molecule. Inhibition of NFAT-mediated transcription by
the dnNFAT resulted in significant suppression of the induction of
NFIL3/E4BP4 mRNA by TG. These data indicate that NFAT is required
for the expression of NFIL3/E4BP4 gene by TG. The NFAT
family has a rel-type DNA-binding domain which lacks sequences in
NF KN-93 prevents the activation of CaM kinase II by interacting with the
calmodulin-binding domain of the kinase (23). As shown in Fig. 6, 1 µM KN-93 markedly reduced the TG-induced NFIL3/E4BP4 expression, whereas 1 µM KN-92, an inactive analog of
KN-93, displayed much less inhibitory effects than KN-93. These results
suggest that CaM kinase signaling is involved in the
TG-dependent NFIL3/E4BP4 induction. Although KN-93 show a
high degree of selectivity for CaM kinase, other nonspecific effects on
cellular function cannot be ruled out. For example, KN-93 suppressed
voltage-dependent K+ channels in vascular
myocytes (41). It is reported that inhibition of
voltage-dependent K+ channels in T lymphocyte
resulted in suppression of calcium signaling and NFAT-driven gene
expression (42). To obtain more direct evidence for the involvement of
CaM kinase signaling, we transfected a CaM kinase II isoform to A7r5
cells. Expression of the CaM kinase II isoform in A7r5 cells resulted
in enhanced elevation of TG-induced NFIL3/E4BP4 expression, relative to
empty vector control expression (data not shown). These data suggest
that CaM kinase signaling is involved in the TG-dependent
NFIL3/E4BP4 induction. Additional studies are required to define that
the CaM kinase responsible for TG-induced NFIL3/E4BP4 expression is CaM
kinase II or other CaM kinases.
CaM kinase II has been reported to mediate the activation of
c-Fos (1), which can form heterodimeric complexes with c-Jun (e.g. AP-1). It is possible that calcineurin and CaM kinase
II activate NFAT and AP-1 expression, respectively, and that NFAT and
AP-1 stimulate the NFIL3/E4BP4 mRNA expression in concert with each
other. Another possibility is that calcineurin and CaM kinase signaling
pathways may act in parallel to preferentially target NFAT and MEF2,
respectively, because CaM kinase I (35) and CaM kinase IV (33-35) can
activate the MEF2 transcription factors. The CaM kinases can also
activate cAMP response element-binding protein and serum response
element-binding protein (1). Therefore, it is possible that these
transcription factors may mediate some of the effects of TG on the
induction of NFIL3/E4BP4 independently of NFAT. CaM kinase II can also
mediate the activation of the ERK pathway following stimulation of
calcium influx by ionomycin in vascular smooth muscle cells (43). Thus,
CaM kinase II may serve to regulate NFIL3/E4BP4 expression via
ERK1/ERK2. In fact, we found that TG-dependent NFIL3/E4BP4
induction was suppressed by pretreatment with 10 µM
PD98059, a specific inhibitor of MEK1/2 (data not shown). In Baf-3
cells, the expression of NFIL3/E4BP4 was regulated by oncogenic Ras
mutant proteins through both the Raf-MAP kinase and
phosphatidylinositol 3-kinase pathways (10). The effect of the MAP
kinase pathway in TG-induced NFIL3/E4BP4 mRNA expression in RASMC
remains to be studied.
In the mouse pro-B cell lines Baf-3 and FL5.12, expression of
NFIL3/E4BP4 was regulated by IL-3 requiring de novo protein synthesis (9). We demonstrated that the protein synthesis inhibitor cycloheximide did not significantly suppress the induction of NFIL3/E4BP4 by TG, suggesting that the mechanism of which IL-3 induces
NFIL3 gene expression in Baf-3 and FL5.12 is different from
the mechanism by which TG induces the expression of NFIL3/E4BP4 mRNA in RASMC. In the human T cell lines MLA144 and HUT78,
NFIL3/E4BP4 mRNA levels increased after PMA stimulation, although
the amount of NFIL3/E4BP4 mRNA did not change following PMA
stimulation in S-LB-1 T cells (8). We found that PMA did not induce the
expression of NFIL3/E4BP4 mRNA in RASMC (data not shown). These
results suggest that there are some differences in the cellular
proteins reacting after PMA stimulation between these cell lines.
It is very important to know the physiological significance of
NFIL3/E4BP4 induction. It is known that NFIL3/E4BP4 functions as an
anti-apoptotic transcription factor in murine
IL-3-dependent Pro-B lymphocytes including Baf-3 and FL5.12
cells (9, 10). In the absence of IL-3, enforced expression of the human
NFIL3/E4BP4 promoted the survival but not the growth of
IL-3-dependent pro-B cells, indicating that induction of
NFIL3/E4BP4 is one of the mechanisms through which IL-3 suppresses
apoptosis (9). The downstream factors through which NFIL3/E4BP4 delays
apoptosis in IL-3-deprived Baf-3 cells have as yet not been identified. One possibility is that NFIL3/E4BP4 induces the expression of cytokine
in Baf-3 cells and blocks apoptosis through an autocrine mechanism,
because this transcription factor has been reported to transactivate
the IL-3 promoter in T cells. We found that rat IL-3
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES
, which is an
essential element that positively regulates the transcription of human
hepatitis B virus genes (7). The potent suppression of box
activity by NFIL3/E4BP4 may contribute to the silencing of
hepatitis B virus gene expression (7).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. The filter was re-probed with
32P-random prime-labeled fragment of the rat
-actin
cDNA to ensure approximately equally loading of RNA samples.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and predicted amino acid sequences
of rat NFIL3/E4BP4 cDNA (accession number AY004663). The amino
acid sequence of rat NFIL3/E4BP4 is shown in the single-letter
code below the nucleotide sequence. The canonical polyadenylation
signal is underlined.
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Fig. 2.
Comparison of the deduced amino acid
sequences of rat, mouse, and human NFIL3/E4BP4. The
asterisks indicate amino acid residues that are identical in
all four sequences. The basic amino acid regions are
underlined and the leucine repeat regions are shown in
italics.
-actin cDNA to ensure equal loading.
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[in a new window]
Fig. 3.
Tissue distribution of rat NFIL3/E4BP4.
Two µg/lane of poly(A)+ RNA, prepared from the indicated
rat tissues, were analyzed by Northern blotting using probes derived
from cDNA for rat NFIL3/E4BP4 (upper panel) or rat
-actin (lower panel).
View larger version (12K):
[in a new window]
Fig. 4.
Effects of thapsigargin on NFIL3/E4BP4
mRNA levels in A7r5 cells. A,
time-dependent effects of thapsigargin on NFIL3/E4BP4
mRNA levels. Serum-starved A7r5 cells were incubated with 300 nM thapsigargin for the indicated periods of time. RNA was
isolated and analyzed by real-time RT-PCR. The results are normalized
for GAPDH mRNA levels and are representative of three similar
experiments performed in triplicate. B,
dose-dependent effects of thapsigargin on NFIL3/E4BP4
mRNA levels. Serum-starved A7r5 cells were treated with the
indicated doses of thapsigargin for 3 h. RNA was isolated, and
analyzed by real time RT-PCR. The results are normalized for GAPDH
mRNA levels and are representative of three similar experiments
performed in triplicate.
View larger version (14K):
[in a new window]
Fig. 5.
Induction of NFIL3/E4BP4 mRNA by
thapsigargin is dependent on elevation of
[Ca2+]i. A, serum-starved A7r5
cells were treated with 300 nM thapsigargin, 100 nM A23187, or 5 µg/ml tunicamycin for 3 h. RNA was
isolated and analyzed by real-time RT-PCR. The results are normalized
for GAPDH mRNA levels and are representative of three similar
experiments performed in triplicate. B, serum-starved A7r5
cells were treated with 300 nM thapsigargin with or without
pretreatment with 2 mM EGTA or 10 µM BAPTA-AM
for 6 h after which total RNA was extracted and analyzed for
NFIL3/E4BP4 mRNA expression by real-time RT-PCR. The results are
normalized for GAPDH mRNA levels and are representative of three
similar experiments performed in triplicate.
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[in a new window]
Fig. 6.
Effect of CsA, FK506, and KN-93 on the
induction of NFIL3/E4BP4 mRNA by thapsigargin. Serum-starved
A7r5 cells were treated with the 30 nM thapsigargin for
3 h with or without pretreatment of 1 µM CsA, 1 µM FK506, 1 µM KN-93, or 1 µM
KN-92. RNA was isolated and analyzed by real-time RT-PCR. The results
are normalized for GAPDH mRNA levels and are representative of
three similar experiments performed in triplicate.
View larger version (17K):
[in a new window]
Fig. 7.
Effect of actinomycin D or cycloheximide on
NFIL3/E4BP4 mRNA induction by thapsigargin. Serum starved
primary A7r5 cells were either left untreated or treated with 300 nM thapsigargin for 3 h with or without pretreatment
with actinomycin D or cycloheximide. Total RNA was extracted and
analyzed for NFIL3/E4BP4 expression by real-time RT-PCR. The results
are normalized for GAPDH mRNA levels and are representative of
three similar experiments performed in triplicate.
View larger version (15K):
[in a new window]
Fig. 8.
A dominant negative form of calcineurin
A2 (CNA2) blocks the induction of
NFIL3/E4BP4 mRNA by thapsigargin. A7r5 cells were transiently
transfected with the control expression vector pcD, or the dominant
negative form of calcineurin pcD-CNA2(H101Q/H290Q), or its wild
type pcD-CNA2. Transfection efficiency was controlled by
co-transfection of the pcD-Luc expression vector. 48 h after
transfection, cells were treated with or without 30 nM
thapsigargin for 3 h and assayed for NFIL3/E4BP4 mRNA levels
by real-time RT-PCR. The results are normalized for luciferase mRNA
levels and are representative of two similar experiments performed in
triplicate.
View larger version (17K):
[in a new window]
Fig. 9.
A dominant negative form of NFAT blocks the
induction of NFIL3/E4BP4 mRNA by thapsigargin. A7r5 cells were
transiently transfected with the control expression vector pcD, or the
dominant negative form of NFAT pcD-NFAT3 (PxIxIT), or its mutant form
pcD-NFAT3 (AxIxIA). Transfection efficiency was controlled by
co-transfection of the pcD-Luc expression vector. 48 h after
transfection, cells were treated with or without 30 nM
thapsigargin for 3 h and assayed for NFIL3/E4BP4 mRNA levels
by real-time RT-PCR. The results are normalized for luciferase mRNA
levels and are representative of two similar experiments performed in
triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B p50 that interact with DNA (28). In addition, a critical
arginine involved in DNA binding by NF
B is substituted with a
histidine in NFAT family members (28). NFAT family members have evolved
to interact weakly with DNA and to require a partner for high-affinity
DNA binding at most sites (28). Complexes induced through other
signaling pathways such as AP-1 (30, 32) or cell type-specific proteins
such as MEF2 (33-35), GATA2 (36), GATA4 (37), or others (38, 39) can
provide the partner. Although the human NFIL3/E4BP4 gene is
mapped to 9q22 (40), the complete genome sequences of human, mouse, and
rat NFIL3/E4BP4 are currently unknown. Homo sapiens
chromosome 9 clone RP11-440G5 (GenBankTM accession number
AL353764) appears to contain the human NFIL3/E4BP4 gene. The
structure of the NFIL3/E4BP4 gene and the exact mechanisms by which NFAT activates the expression of NFIL3/E4BP4 are still to be determined.
mRNA (44)
was induced by TG in RASMC (data not shown). Although NFAT can directly
activate the human GM-CSF/IL-3 promoter (28, 45) and IL-3 mRNA is
stabilized following stimulation of calcium influx by ionomycin (46),
it is also possible that TG might activate IL-3
expression through
the induction of NFIL3/E4BP4 expression in RASMC. The induction of
NFIL3/E4BP4 by calcium signaling might have some critical functions in
apoptosis in vascular smooth muscle cells.
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ACKNOWLEDGEMENTS |
---|
We thank Hai An Zheng for assistance with experiments and the members of the Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, for their continual help and support.
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FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for Scientific Research on Priority Areas, Scientific Research (A) and (B), International Scientific Research (Joint Research) and Exploratory Research from the Ministry of Education, Science, Sports and Culture, a grant from the Pediatric Diseases from the Ministry of Health and Welfare, the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research, Japan, grants for Research Projects on Muscle Regulation and on Cerebral Vasospasm from Mie University School of Medicine, and a Grant-in-Aid 1996 from the Mie Medical Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) data of rat NFIL3/E4BP4 reported in this paper has been submitted to DDBJ, EMBL, and NCBI data bases with the accession number(s) AY004663.
To whom correspondence should be addressed: Dept. of Molecular and
Cellular Pharmacology, Mie University School of Medicine, Edobashi,
Tsu, Mie 514-8507, Japan. Tel.: 81-59-232-5006; Fax: 81-59-232-1765;
E-mail: tanaka@doc.medic.mie-u.ac.jp.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M010332200
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ABBREVIATIONS |
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The abbreviations used are: TG, thapsigargin; NFAT, nuclear factor of activated T cells; RASMC, rat aortic smooth muscle cells; PMA, phorbol 12-myristate 13-acetate; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-polymerase chain reaction; aa, amino acid(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ER, endoplasmic reticulum; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl; CsA, cyclosporin A; dnNFAT, dominant-negative nuclear factor of activated T cells; ERK, extracellular regulated kinase; NFIL-3, nuclear factor regulated by interleukin 3; E4BP4, adenovirus E4 binding protein; CaM, calmodulin; CaM kinase, calcium/calmodulin-dependent protein kinase.
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