Pulmonary and Critical Care Medicine, Stanford University Medical Center, Stanford, California 94305-5236
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human bronchial epithelial (HBE) cells express interleukin (IL)-2 [Y. Aoki, D. Qiu, A. Uyei, and P. N. Kao. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L276-L286, 1997]. 16HBE-transformed cells contain constitutive and inducible nuclear DNA-binding activity for the purine-box/nuclear factor (NF) of activated T cell (NFAT) target DNA sequence in the human IL-2 enhancer. Transcriptional activation through the purine-box DNA sequence requires stimulation with phorbol 12-myristate 13-acetate + ionomycin, and this activation is inhibited by cyclosporin A. Immunohistochemical staining of 16HBE cells demonstrates nuclear expression of the purine-box DNA-binding proteins NF45 and NF90 and no expression of NFATp or NFATc. NF90 and NF45 associate with the DNA-dependent protein kinase catalytic subunit and the DNA-targeting subunits Ku80 and Ku70 (N. S. Ting, P. N. Kao, D. W. Chan, L. G. Lintott, and S. P. Lees-Miller. J. Biol. Chem. 273: 2136-2145, 1998). Antibodies to Ku potently inhibit the purine-box DNA-binding complex. The purine-box transcriptional regulator in 16HBE cells likely comprises NF45, NF90, Ku80, Ku70, and the DNA-dependent protein kinase catalytic subunit.
cyclosporin A; deoxyribonucleic acid-dependent protein kinase; nuclear factor of activated T cells; interleukin-2
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BRONCHIAL EPITHELIAL CELLS exist at the interface between the host and the external environment and initiate and regulate the airway inflammatory response to noxious stimuli and pathogens. Inflammatory signaling molecules expressed by human bronchial epithelial (HBE) cells include the cytokines tumor necrosis factor, interleukin (IL)-1, IL-6, and IL-8 (reviewed in Ref. 30); leukotrienes (4); and hematopoietic growth factors including granulocyte-macrophage colony-stimulating factor (GM-CSF) (10) and IL-2 (3). The production of hematopoietic growth factors by bronchial epithelium likely promotes the local proliferation and functional maturation of immune effector cells recruited from the circulation into the microenvironment of the airways. This ability of bronchial epithelial cells to modulate the development of a local immune response is important for effective host defense against foreign pathogens. However, excessive growth factor expression by bronchial epithelial cells likely contributes to the cellular inflammation that is characteristic of noninfectious airway inflammatory conditions such as asthma (6, 15).
Aoki et al. (3) were the first to demonstrate that HBE
cells express IL-2 mRNA and protein. IL-2 is expressed primarily by
activated T cells and is regulated at the level of transcription. In
activated T cells, transcriptional regulation at the IL-2 enhancer involves binding and activation of the specific transcription factors nuclear factor (NF) of activated T cells (NFAT), NF-B, octamer-1, and activator protein-1 (AP-1) (reviewed in Ref.
28). The target DNA sequence for NFAT has a purine-rich core motif, GAGGAAAA, which has been designated as the purine-box (reviewed in Ref.
28) or, alternatively, the antigen-receptor response element (ARRE)
(29). Multimerized purine-box/ARRE/NFAT target DNA elements confer
stimulation-dependent and cyclosporin A (CsA)-sensitive transcriptional
activation on a linked reporter gene transfected into T cells (17).
Purine-box/ARRE DNA-binding activity and regulated transcriptional
activation have been shown to exist in T cells, B cells (33), and
endothelial cells (11). Purine-box transcriptional regulatory elements
that are inhibited by CsA have also been identified in the promoters of
the IL-3, IL-4, GM-CSF, and tumor necrosis factor-
genes (12, 27,
28) .
Nuclear proteins that bind specifically to the purine-box in the IL-2 enhancer have been identified and cloned by diverse laboratories. Corthesy and Kao (13) and Kao et al. (19) purified a purine-box/ARRE DNA-binding complex from the nuclei of activated Jurkat T cells and identified and cloned proteins NF45 and NF90. NFATp and NFATc proteins were isolated from the cytoplasms of T cells and are proposed to translocate into the nucleus and combine with the AP-1 proteins Fos and Jun to activate transcription at the purine-box/NFAT target DNA sequence (27; reviewed in Ref. 28).
Ting et al. (31) recently showed that NF45 and NF90 associate tightly with DNA-dependent protein kinase (DNA-PK). DNA-PK consists of a large catalytic subunit, DNA-PKcs, and DNA-targeting subunits Ku80 and Ku70 (20). DNA-PK is involved in double-strand DNA break repair and V(D)J recombination (9, 20). The association of DNA-PK with an RNA polymerase II complex suggests a role for DNA-PK in the regulation of transcription (21). Sequence-specific transcriptional repression has been shown to be mediated by DNA-PKcs and specific binding of Ku to a purine-rich target DNA-sequence (18). In vitro reconstitution of NF90, NF45, Ku80, Ku70, and DNA-PKcs leads to the formation of a large protein complex that binds to DNA in an electrophoretic mobility shift assay (EMSA) (31).
Here we characterize, for the first time, a purine-box transcriptional regulator complex that exists constitutively in the nucleus of 16HBE cells. The purine-box regulator binds to its target DNA sequence in nonstimulated 16HBE cells and is transcriptionally silent. Stimulation of 16HBE cells increases purine-box DNA-binding activity, activates transcription, and induces IL-2 protein secretion. Purine-box DNA-binding activity, transcriptional activation, and IL-2 expression are inhibited by the T-cell immunosuppressants CsA and FK506. We performed immunohistochemistry on 16HBE cells and demonstrate that NF45 and NF90 are strongly expressed constitutively in the nucleus and that NFATp and NFATc are not expressed at all. NF90 and NF45 associate with DNA-PKcs and Ku, and we show that antibodies against Ku potently inhibit the 16HBE cell purine-box DNA-binding complex. We infer that the purine-box transcriptional regulator in 16HBE cells consists of NF90, NF45, Ku80, Ku70, and DNA-PKcs.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and stimulation
conditions. An SV40 large T antigen-transformed HBE
cell line 16HBE14o (16HBE cells), which retains differentiated
morphology and function of normal human airway epithelia (14), was
cultured in Eagle's minimum essential medium (BioWhittaker,
Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine
serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin
(BioWhittaker) as previously described (3). An adult human T-cell
leukemic cell line, Jurkat (clone E6-1), was obtained from American
Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium
(Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum,
penicillin, and streptomycin (BioWhittaker). Monolayer epithelial cells
grown to 90% confluency or Jurkat T cells (~1 × 106 cells/ml) were stimulated for
the indicated period of time in culture medium containing 20 ng/ml of
phorbol 12-myristate 13-acetate (PMA; Calbiochem, La Jolla, CA) or PMA
plus 2 µM ionomycin (Iono; Calbiochem). Modulation of 16HBE cell
purine-box DNA-binding activity and transcriptional induction was
investigated in response to CsA (Sandoz Research Institute, East
Hanover, NJ), dibutyryl cAMP (Calbiochem),
PGE2, theophylline, isoproterenol,
and erythromycin (Sigma, St. Louis, MO). In these experiments, cells
were pretreated with each drug for 16 h before stimulation with PMA or
Iono for 2 h in the continued presence of each drug, followed by
preparation of bronchial epithelial cell nuclear or whole cell extracts.
Nuclear extract preparation and EMSAs.
The 16HBE and Jurkat T cells were stimulated for 2 h, pelleted in
microfuge tubes, and then rinsed with cold phosphate-buffered saline
(PBS). The nuclear extracts were prepared on ice with ice-cold reagents
at 4°C as previously described (13). The cell pellets were
resuspended with 300 µl of buffer A
(10 mM HEPES, pH 7.6, 15 mM KCl, 2 mM MgCl2, and 0.1 mM EDTA, pH 8.0)
containing the following supplements: 1 mM dithiothreitol, 0.2 (16HBE)
or 0.05% (Jurkat) Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl
fluoride, 1 mM NaVO3, 10 mM NaF, 1 mM sodium pyrophosphate, 5 mM benzamidine, 0.1 mM sodium molybdate, and
1 mM -glycerophosphate. After a 10-min incubation on ice, nuclei
were spun down at 4,000 rpm for 5 min and were then resuspended in one
volume of buffer C (25 mM HEPES, pH
7.6, 50 mM KCl, 0.1 mM EDTA, pH 8.0, and 10% glycerol) containing the supplements except NP-40. DNA-binding proteins were extracted from
chromatin by incubating the nuclear suspension with a one-ninth volume
of 3 M
(NH4)2SO4
for 30 min on ice, followed by centrifugation and pelleting of
chromatin at 75,000 rpm for 15 min. The nuclear proteins extracted into
the supernatant were precipitated with one volume of 3 M
(NH4)SO4
for 10 min on ice, followed by ultracentrifugation at 50,000 rpm for 10 min. The pellet of precipitated nuclear proteins was carefully
resuspended with 100 µl of buffer C
containing the supplements and dialyzed against 100 ml of
buffer C without the supplements for 3 h at 4°C. The concentration of nuclear proteins was measured with
the Bradford dye-binding assay (Bio-Rad, Hercules, CA).
Purine-box DNA-binding activity in 16HBE or Jurkat nuclear extracts was
assayed by EMSA. Briefly, 5-10 µg of nuclear proteins were
incubated for 40 min at 4°C in 20 µl of binding buffer (25 mM
HEPES, pH 7.6, 0.1 mM EDTA, 10% glycerol, 50 mM KCl, and 0.05 mM
dithiothreitol) containing 1 µg of poly(dI-dC) and 2.5 pg of 32P-labeled oligonucleotide probe
(~1 × 105 counts/min) for
the purine-box/NFAT sequence in the human IL-2 enhancer (bases
259 to
284; upper strand,
5'-AAGAAAGGAGGAAAAACTGTTTCATA-3'; lower strand,
5'-TCTGTATGAAACAGTTTTTCCTCCTT-3') (28). The probe was
labeled with Klenow DNA polymerase (New England Biolabs, Beverly, MA)
to fill in the four-base overhangs on each end of the annealed strands
with [
-32P]dCTP
(Amersham, Arlington Heights, IL) and nonradioactive dATP, dTTP, and
dGTP. The competitor oligonucleotides utilized were the purine-box/NFAT
sequence in the human GM-CSF gene (GM-550; 5'-agctGAAAGGAGGAAAGCAAGAGTCATA-3') (12), the NF-
B
sequence from the mouse Ig
-light chain (Ig NF-
B;
5'-agctAAA
-3') (5),
and the Sp1 sequence
(5'-agctGA
GAGC-3').
The lowercase segment agct represents an appended sequence that
overhangs the annealed strands and is available for fill-in
radiolabeling; the underlined segments indicate the target DNA-binding
sequences for NF-
B and Sp1, respectively. Protein-DNA complexes were
resolved from free probe on 4% nondenaturing polyacrylamide gels in
0.5× Tris-borate-EDTA (pH 8.3) and visualized by fluorography.
Cell transfections and luciferase reporter gene
assays. Two independent 16HBE cell lines carrying the
purine-box luciferase reporter gene construct were generated according
to the methods previously described (3). The purine-box reporter
construct contains three copies of the distal purine-box in the human
IL-2 enhancer (bases 255 to
285; NFAT target DNA
sequence) in the context of the minimal IL-2 promoter, driving the
firefly luciferase cDNA (16). The reporter construct also contains a
neomycin resistance gene driven by the SV40 enhancer. Cells were
stimulated for the indicated times, and whole cell extracts were
prepared and assayed for luciferase activity as previously described
(3). A 16HBE cell line was generated with the EF-1
promoter (32)
driving expression of histidine-tagged NF45 protein (19). There were no
significant differences in the expression of NF45 or in the purine-box
EMSA between NF45-transfected cells versus nontransfected 16HBE cells
(data not shown).
Immunohistochemistry and Western immunoblotting. Adherent 16HBE cells were harvested by trypsinization, washed, and resuspended in fresh medium, and one drop of the cell suspension was spotted onto a bovine serum albumin-coated diamond pen-scribed microscopy slide and incubated overnight in a moisture box at 37°C and 5% CO2. The cells were fixed over 15 min at 4°C with 4% paraformaldehyde in PBS, then washed twice in PBS. Fixed cells were exposed to blocking buffer (2% sucrose and 0.1 M glycine in PBS) for 30 min, dehydrated in 100% methanol, permeablized with 3% hydrogen peroxide in PBS for 5 min at room temperature, and then rinsed in PBS. Primary antibodies were added in PBS as purified rabbit IgGs (NF45 and NF90) at 4 µg/ml or goat IgGs [NFATp (M-20) and NFATc (K-18)] at 2 µg/ml according to the manufacturer's instructions (Santa Cruz Biotechnology, Santa Cruz, CA) and incubated in the humidified box at room temperature for 30 min, and then the slides were rinsed in PBS. Detection was incubation with the appropriate biotinylated anti-rabbit or anti-goat secondary antibody (1:400 dilution; Jackson Immunoresearch, West Grove, PA) for 30 min and rinsing with PBS, followed by incubation with streptavidin-horseradish peroxidase (1:400; Jackson Immunoresearch) for 30 min and rinsing with PBS, and then staining with diaminobenzidine (2.5%) for 5 min. The cells were counterstained with hematoxylin for 30 s. Rabbit antisera to NF45 and NF90 were generated by BABCO (Berkeley, CA) with amino-terminal histidine-tagged NF45 (amino acids 1-194) and amino-terminal histidine-tagged NF90 (amino acids 1-151) as immunogens. The NF45 and NF90 IgGs were purified from the immune sera with protein A-agarose (Zymed, South San Francisco, CA). For immunoblotting, nuclear proteins were fractionated by SDS-PAGE (8% separating gel) and transferred electrophoretically to nitrocellulose. Primary incubations utilized antibodies 111 and N3H10 (monoclonal anti-Ku80 and -Ku70, respectively; provided by W. Reeves, University of North Carolina, Chapel Hill), rabbit polyclonal anti-DNA-PKcs (DPK1; provided by S. Lees-Miller, University of Calgary, Calgary, AL), or anti-NF45 and anti-NF90 (rabbit polyclonal sera). Detection was with horseradish peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence (Amersham).
Data and statistical analysis. Significance of the differences between the experimental conditions was determined by paired two-sample Student's t-test (Microsoft EXCEL).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aoki et al. (3) previously showed that normal and transformed HBE cells express the T-cell growth factor IL-2. Here we characterize the DNA-binding properties, transcriptional regulation, sensitivity to immunomodulating drugs, and subunit composition of a purine-box transcriptional regulator involved in 16HBE cell expression of IL-2.
Characterization of CsA- and FK506-sensitive purine-box DNA-binding activity in the nucleus of 16HBE cells. We prepared nuclear extracts from 16HBE and Jurkat T cells and compared the recovery of purine-box DNA-binding activity. In EMSAs, we used a 32P-labeled oligonucleotide probe that corresponds exactly to the purine-box/NFAT target DNA sequence from the human IL-2 enhancer. It is apparent that 16HBE cells contain a nuclear protein complex that binds specifically to the purine-box/NFAT oligonucleotide probe (Fig. 1A, lanes 1-3) and migrates with the identical electrophoretic mobility as the complex in the nucleus of Jurkat T cells, which is known as NFAT (Fig. 1B, lanes 2 and 3). In nonstimulated 16HBE cells (Fig. 1A, lane 1) but not in nonstimulated Jurkat T cells (Fig. 1B, lane 1), there is substantial purine-box DNA-binding activity. In 16HBE cells, there is a modest induction of the purine-box DNA-binding complex after stimulation with PMA and PMA+Iono (Fig. 1A, lanes 2 and 3 vs. lane 1); in Jurkat T cells, purine-box/NFAT DNA-binding activity is substantially induced with PMA alone and even more potently induced after stimulation with PMA+Iono (Fig. 1B, lanes 2 and 3 vs. lane 1). In both 16HBE and Jurkat T cells, CsA destabilizes the purine-box/NFAT DNA-binding complex (Fig. 1, A and B, lanes 4-6). These data demonstrate that 16HBE cells contain a nuclear purine-box DNA-binding activity that is induced with PMA+Iono and inhibited by CsA. The 16HBE cell purine-box DNA-binding complex has similar electrophoretic mobility, induction characteristics, and CsA sensitivity as the Jurkat T cell NFAT complex.
|
The 16HBE cell purine-box DNA-binding complex that is induced by PMA+Iono is also potently inhibited by the T-cell immunosuppressant FK506 (Fig. 1C, lanes 4-6). In the presence of 1 µM FK506, the PMA+Iono-stimulated purine-box DNA-binding complex is inhibited to a level below that present in nonstimulated nuclear extracts (Fig. 1C, lane 6 vs. lane 1). The 16HBE cell purine-box DNA-binding complex is inhibited by 1 µM CsA (Fig. 1D, lane 3) and 0.1 and 1 µM FK506 (Fig. 1E, lanes 2 and 3) in the absence of any calcium signaling.
Transcriptional activation through the purine-box DNA sequence in 16HBE cells. After we established that 16HBE cells contain a nuclear protein complex that binds specifically to the purine-box/NFAT sequence and migrates with similar electrophoretic mobility as the complex designated NFAT in T cells, we next investigated whether 16HBE cells show regulated transcriptional activation of a purine-box/NFAT luciferase reporter gene. We generated 16HBE cells that stably express the purine-box/NFAT luciferase reporter gene and evaluated the stimulation requirements for transcriptional activation. It is apparent that there is no transcriptional activation of the reporter construct in nonstimulated or PMA-stimulated 16HBE cells (Fig. 2A), and significant transcriptional activation of the purine-box/NFAT reporter in 16HBE cells is achieved after stimulation with PMA+Iono (Fig. 2A). These induction requirements in 16HBE cells mimic exactly the induction requirements for NFAT transcriptional activation in T cells (28). The results observed in two independent 16HBE cell lines that stably express the purine-box/NFAT luciferase reporter are similar to the results observed in transiently transfected 16HBE cells (Fig. 2A).
|
We next examined the effects of CsA on transcriptional activation of the purine-box/NFAT luciferase reporter in 16HBE cells stimulated with PMA+Iono (Fig. 2B). It is apparent that CsA causes a dose-dependent inhibition in purine-box transcriptional activation in 16HBE cells. The half-maximal effective dose for transcriptional inhibition by CsA in 16HBE cells is ~4 ng/ml (Fig. 2B), and this is the same half-maximal effective dose as for CsA inhibition of NFAT transcription in Jurkat T cells (data not shown).
Effects of CsA on 16HBE cell expression of IL-2. We next investigated whether 16HBE cell expression of IL-2 is regulated by stimulation and CsA similar to the activation of the purine-box transcriptional regulator (Fig. 3). It is apparent that 16HBE cell expression of IL-2 protein requires stimulation with PMA+Iono (Fig. 3), and this is similar to the requirement for PMA+Iono stimulation for transcriptional activation of the purine-box/NFAT luciferase reporter (Fig. 2A). Furthermore, 16HBE cell expression of IL-2 protein stimulated by PMA+Iono is completely inhibited by CsA (1,000 ng/ml; Fig. 3), and this is similar to the inhibition of purine-box/NFAT luciferase activity by CsA (Fig. 2B). The similarities in induction requirements and sensitivity to inhibition by CsA of the purine-box transcriptional regulator and in 16HBE cell expression of IL-2 protein imply that the CsA-sensitive purine-box transcriptional regulator is involved in 16HBE cell expression of IL-2.
|
Effects of drugs on 16HBE cell purine-box regulator induction and transcriptional activation. We next investigated the effects of other immunomodulating drugs on the induction of the 16HBE cell purine-box regulator DNA-binding complex and on transcriptional activation of the purine-box/NFAT luciferase reporter (Fig. 4). By EMSA, it is apparent that dibutyryl cAMP and isoproterenol showed minimal inhibition; PGE2, theophylline, and erythromycin showed moderate inhibition; and CsA showed complete inhibition of the purine-box DNA-binding complex (Fig. 4, A and B). In the purine-box/NFAT luciferase reporter assay (Fig. 4C), these drugs showed inhibitory effects that corresponded well to their effects on the purine-box DNA-binding activity (compare Fig. 4C with Fig. 4, A and B). After CsA, the most potent inhibitors of purine-box/NFAT-luciferase activity are PGE2, theophylline, and erythromycin.
|
Immunological analysis of candidate purine-box transcriptional regulator subunits. To identify subunits of the purine-box transcriptional regulator in 16HBE cells, we used antibodies to investigate the expression and subcellular localization of proteins potentially involved in specific binding to purine-box/NFAT target DNA sequence. We performed immunoperoxidase staining of 16HBE cells and demonstrated constitutive nuclear expression of the proteins NF45 and NF90 (Fig. 5A). In 16HBE cells undergoing mitosis, we observed that the pattern of immunoreactivity for NF45 and NF90 moves from the nucleus into the cytoplasm (Fig. 5A). There are no signficant changes in the level or subcellular localization of NF45 or NF90 immunoreactivity after 16HBE cell stimulation or treatment with CsA (data not shown).
|
In contrast, we observed no immunoreactivity for any NFATp or NFATc proteins detectable in resting or stimulated 16HBE cells (Fig. 5A and data not shown). In particular, the NFATc (K-18) antiserum, described as broadly reactive against NFAT family members, showed nuclear immunoreactivity in Jurkat T cells (data not shown) but showed no immunoreactivity in 16HBE cells (Fig. 5A). These results imply that the purine-box regulator in 16HBE cells does not contain any NFATp or NFATc family polypeptides. The strong expression of NF45 and NF90 proteins in the nucleus of 16HBE cells is consistent with the hypothesis that NF45 and NF90 proteins, but not NFATp or NFATc proteins, contribute to 16HBE cell purine-box DNA-binding activity and transcriptional activation.
By Western immunoblotting, we show that nuclear extracts prepared from 16HBE cells demonstrate substantial expression of NF45, NF90, and DNA-PK subunits Ku80, Ku70 and DNA-PKcs (Fig. 5B). There was no detectable immunoreactivity with the broadly reactive NFATc antiserum (data not shown).
Oligonucleotide competitions and Ku antibody
inhibition of specific 16HBE cell purine-box DNA-binding
activity. To investigate the specific binding of the
purine-box regulator complex in 16HBE cells, we performed competitions
with unlabeled oligonucleotides related to, and distinct from, the IL-2
purine-box/NFAT target DNA sequence (Fig.
6A).
There is substantial purine-box DNA-binding activity in the nucleus of
nonstimulated 16HBE cells, which is further enhanced after stimulation
with PMA+Iono (Fig. 6A,
lanes 1 and
2). The purine-box regulator
DNA-binding complex in PMA+Iono-stimulated 16HBE cells is strongly
inhibited by the self purine-box/NFAT oligonucleotide
(Fig. 6A, lane
3) and is partially inhibited by the related GM-CSF
promoter NFAT site oligonucleotide (Fig.
6A, lane
4). The mouse Ig NF-B sequence oligonucleotide
also shows partial inhibition of the specific purine-box regulator
complex (Fig. 6A,
lane 5), and the unrelated Sp1
oligonucleotide shows essentially no competition for binding to the
purine-box regulator complex (Fig. 6A,
lane 6). These oligonucleotide
competition experiments establish that the 16HBE cell purine-box
regulator complex binds with sequence specificity to the
purine-box/IL-2 NFAT DNA target sequence.
|
Ting et al. (31) previously showed that NF45 and NF90 proteins
associate with the DNA-PKcs and the DNA-targeting subunits Ku70 and
Ku80, and we hypothesized that the purine-box transcriptional regulator
in 16HBE cells comprises NF90, NF45, Ku80, Ku70, and DNA-PKcs. To test
this hypothesis, we investigated the effects of monoclonal antibodies
against Ku80 and Ku70 (34) on the specific purine-box DNA-binding
complex in 16HBE cell nuclear extracts (Fig.
6B). We found that antibody 162, which recognizes native Ku70 and Ku80, substantially inhibits the
purine-box DNA-binding complex (Fig.
6B, lane
3 vs. lane 2).
Antibody 111, which recognizes human Ku80, causes complete inhibition
of the purine-box DNA-binding complex in stimulated 16HBE cells (Fig.
6B, lane
4 vs. lane 2). Antibody N3H10, which recognizes human Ku70, causes near-complete inhibition of the purine-box DNA-binding complex (Fig.
6B, lane 5 vs. lane 2). As a
control, we show that the polyclonal rabbit IgG directed against
cytoplasmic phospholipase A2 shows
no significant inhibition of the purine-box DNA-binding complex (Fig.
6B, lane 6 vs. lane 2). The
monoclonal antibodies against Ku produce no significant inhibition of
the Ig NF-kB complex in T cells (data not shown), establishing that
the Ku antibody inhibition of the purine-box DNA-binding complex in
16HBE cells is specific.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have characterized the DNA-binding properties, induction requirements for transcriptional activation, and subunit composition of a purine-box transcriptional regulator that controls the expression of IL-2 by 16HBE cells.
Previously, Corthesy and Kao (13) and Kao et al. (19) isolated a purine-box DNA-binding complex from nuclear extracts of Jurkat T cells and identified and cloned the polypeptides NF45 and NF90. During the purification, Ku70 and Ku80 were isolated as components of the intact T-cell purine-box transcriptional regulator complex (Kao, unpublished data). NF45 and NF90 are proteins of novel structure: NF45 has sequence similarity to helicases and NF90 contains two domains predicted to bind double-stranded RNA. NF45 and NF90 proteins are localized predominantly in the nucleus of all cell types examined (19). Recently, Ting et al. (31) identified NF45 and NF90 as copurifying polypeptides tightly associated with DNA-dependent protein kinase. In collaboration with Ting et al., we showed that NF45 and NF90 proteins associate with the DNA-PKcs and Ku70 and Ku80 to form a large multisubunit DNA-binding complex.
The purine-box DNA-binding complex that we have characterized in 16HBE cells migrates with identical electrophoretic mobility as the complex defined as NFAT in nuclear extracts prepared from stimulated Jurkat T cells. Furthermore, the 16HBE cell purine-box regulator complex and the Jurkat T cell purine-box/NFAT complex are each induced after stimulation with PMA+Iono and are each inhibited by CsA and FK506. In contrast to the situation in T cells, 16HBE cells express substantial nuclear purine-box DNA-binding activity in resting conditions. This purine-box DNA-binding activity in 16HBE cells is destabilized by CsA and FK506 in the absence of any calcium signaling. This result implies that CsA and FK506 can inhibit the purine-box transcriptional regulator through mechanisms that do not involve calcium signaling or calcineurin. Such calcium-independent effects of CsA were demonstrated in a mast cell line, in which CsA was shown to destablize IL-3 mRNA (26). These authors suggested that CsA inhibits the expression of an RNA-stabilizing protein, without which IL-3 mRNA is rapidly degraded.
We hypothesized that the purine-box transcriptional regulator in 16HBE cells switches from a transcriptional repressor into a transcriptional activator on cell stimulation with PMA+Iono. 16HBE cells that are nonstimulated or stimulated with PMA alone show substantial purine-box DNA-binding activity, yet purine-box luciferase transcriptional activation is silent. Only after stimulation with PMA+Iono is there a modest enhancement in DNA binding, which is associated with significant transcriptional activation. We propose that the transcriptionally silent form of the purine-box DNA-binding complex is a sequence-specific transcriptional repressor in 16HBE cells. In T cells, the purine-box/NFAT target DNA sequence has been shown to be involved in sequence-specific transcriptional repression as well as transcriptional activation (24, 25). We show in 16HBE cells that stimulation with PMA+Iono enhances the purine-box DNA-binding complex, is associated with transcriptional activation of the purine-box luciferase reporter construct, and, importantly, induces expression of IL-2 protein. Because stimulation that triggers transcriptional activation and IL-2 expression is associated with an increased intensity of the purine-box DNA-binding complex, we believe that there is not a replacement of the repressor with a different activator but, rather, a conversion of the DNA-bound purine-box regulator complex from a repressor into an activator.
CsA and FK506 act to destabilize the purine-box regulator complex in
16HBE cells. In contrast to T cells, we can observe this destabilization effect of CsA and FK506 on the purine-box complex in
the absence of stimulation with PMA+Iono. If the purine-box regulator
can mediate transcriptional repression as we propose, then we predict
that CsA will inhibit repression or derepress transcriptional
activation. Others (8) have described that there can be cross
competitition between transcriptional regulators of the NFAT and
NF-B target sequences. Aoki and Kao (2) have previously shown that
CsA can inhibit calcium-mediated repression of NF-
B activity in
16HBE cells. Our results established a mechanism through which CsA, by
destabilizing a repressor, can exert proinflammatory effects in
nonlymphoid cells.
We evaluated the effects of immunomodulating drugs on transcriptional activation of the purine-box regulator in 16HBE cells. Purine-box transcription that is stimulated with PMA+Iono is potently inhibited by CsA and is partially inhibited by PGE2 and theophylline. PGE2 has been shown to exert anti-inflammatory effects in T cells by inhibiting T-cell activation (1). The mechanisms of PGE2 anti-inflammatory effects may partially involve elevation of intracellular cAMP (23); however, because the inhibition of 16HBE cell purine-box transactivation observed with PGE2 was much more profound than that with dibutryl cAMP, it is likely that PGE2 mechanisms involve other pathways beyond elevation of intracellular cAMP. Similarly, theophylline, which can lead to increases in intracellular cAMP, showed substantially more inhibition of purine-box transactivation than did dibutryl cAMP, suggesting again that the anti-inflammatory effects of theophylline (7) likely involve mechanisms beyond the elevation of intracellular cAMP.
Having established that 16HBE cells express a purine-box transcriptional regulator in the nucleus, we sought to identify candidate polypeptides of the purine-box regulator. We performed immunohistochemical experiments and observed strong constitutive expression of the polypeptides NF45 and NF90. NF45 and NF90 polypeptides were detected in the nucleus of nonmitotic 16HBE cells. During mitosis, NF45 and NF90 translocate to the cytoplasm. Similar results have been described for mitotic-phase phosphoprotein 4, which shows an ~90% sequence identity to NF90 (22). The biological significance of this nuclear to cytoplasmic translocation during mitosis is unknown. There was no detectable reactivity for NFATp- or NFATc-related polypeptides with antisera that are both broadly and specifically reactive with NFAT family members.
NF45 and NF90 proteins associate tightly with the DNA-PKcs and stabilize the interaction of the DNA-PKcs with DNA-targeting subunits Ku80 and Ku70 (31). Ku70 and Ku80 proteins bind specifically to purine-rich DNA sequences (18) and interact with DNA-PKcs to mediate sequence-specific transcriptional repression (18). DNA-PKcs and Ku are found in association with the RNA polymerase II complex (21). We have demonstrated that monoclonal antibodies to Ku80 and Ku70 potently and specifically inhibit the purine-box DNA-binding complex in nuclear extracts of stimulated 16HBE cells.
In summary, we have established that human bronchial epithelial cells express a nuclear complex that binds with high affinity and specificity to a purine-box sequence that is identical to the NFAT target DNA sequence in the human IL-2 enhancer. This purine-box regulator complex activates transcription in bronchial epithelial cells in a CsA-inhibitable manner. CsA and FK506 inhibit the DNA-binding and transcriptional activation of this purine-box regulator complex and also inhibit 16HBE cell expression of IL-2. Monoclonal antibodies against Ku80 and Ku70 potently and specifically inhibit the purine-box DNA-binding complex in 16HBE cells. We propose that the purine-box transcriptional regulator in 16HBE cells consists of NF45, NF90, Ku80, Ku70, and the DNA-PKcs and that this complex switches from a transcriptional repressor into a transcriptional activator in response to cell stimulation.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by grants from the California Affiliate of the American Lung Association and the Donald E. and Delia B. Baxter Foundation and by National Institute of Allergy and Infectious Diseases Grants K04-AI-01147 and R01-AI-39624 to P. N. Kao.
![]() |
FOOTNOTES |
---|
Y. Aoki received salary support from Saga Medical School, Saga, Japan.
Present address of Y. Aoki: Dept. of Internal Medicine, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan.
Address reprint requests to P. N. Kao.
Received 22 December 1997; accepted in final form 27 August 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anastassiou, E. D.,
F. Paliogianni,
J. P. Balow,
H. Yamada,
and
D. T. Boumpas.
Prostaglandin E2 and other cyclic AMP-elevating agents modulate IL-2 and IL-2R alpha gene expression at multiple levels.
J. Immunol.
148:
2845-2852,
1992
2.
Aoki, Y.,
and
P. N. Kao.
Cyclosporin A-sensitive calcium signaling represses NFkappaB activation in human bronchial epithelial cells and enhances NFkappaB activation in Jurkat T-cells.
Biochem. Biophys. Res. Commun.
234:
424-431,
1997[Medline].
3.
Aoki, Y.,
D. Qiu,
A. Uyei,
and
P. N. Kao.
Human airway epithelial cells express interleukin-2 in vitro.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L276-L286,
1997
4.
Aoki, Y.,
D. Qiu,
G. H. Zhao,
and
P. N. Kao.
Leukotriene B4 mediates histamine induction of NF-B and IL-8 in human bronchial epithelial cells.
Am. J. Physiol.
274 (Lung Cell. Mol. Physiol. 18):
L1030-L1039,
1998
5.
Baeuerle, P. A.,
and
T. Henkel.
Function and activation of NF-kappa B in the immune system.
Annu. Rev. Immunol.
12:
141-179,
1994[Medline].
6.
Barnes, P. J.
Cytokines as mediators of chronic asthma.
Am. J. Respir. Crit. Care Med.
150:
S42-S49,
1994[Medline].
7.
Barnes, P. J.,
and
R. A. Pauwels.
Theophylline in the management of asthma: time for reappraisal?
Eur. Respir. J.
7:
579-595,
1994
8.
Casolaro, V.,
S. N. Georas,
Z. Song,
I. D. Zubkoff,
S. A. Abdulkadir,
D. Thanos,
and
S. J. Ono.
Inhibition of NFAT-dependent transcription by NF-kappa B: implications for differential gene expression in T helper cell subsets.
Proc. Natl. Acad. Sci. USA
92:
11623-11627,
1995[Abstract].
9.
Chu, G.
Double strand break repair.
J. Biol. Chem.
272:
24097-24100,
1997
10.
Churchill, L.,
B. Friedman,
R. P. Schleimer,
and
D. Proud.
Production of granulocyte-macrophage colony-stimulating factor by cultured human tracheal epithelial cells.
Immunology
75:
189-195,
1992[Medline].
11.
Cockerill, G. W.,
A. G. Bert,
G. R. Ryan,
J. R. Gamble,
M. A. Vadas,
and
P. N. Cockerill.
Regulation of granulocyte-macrophage colony-stimulating factor and E-selectin expression in endothelial cells by cyclosporin A and the T-cell transcription factor NFAT.
Blood
86:
2689-2698,
1995
12.
Cockerill, P. N.,
M. F. Shannon,
A. G. Bert,
and
G. R. Ryan.
The granulocyte-macrophage colony-stimulating factor/interleukin 3 locus is regulated by an inducible cyclosporin A-sensitive enhancer.
Proc. Natl. Acad. Sci. USA
90:
2466-2470,
1993[Abstract].
13.
Corthesy, B.,
and
P. N. Kao.
Purification by DNA affinity chromatography of two polypeptides that contact the NFAT DNA binding site in the interleukin 2 promoter.
J. Biol. Chem.
269:
20682-20690,
1994
14.
Cozens, A. L.,
M. J. Yezzi,
K. Kunzelmann,
T. Ohrui,
L. Chin,
K. Eng,
W. E. Finkbeiner,
J. H. Widdicombe,
and
D. C. Gruenert.
CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.
Am. J. Respir. Crit. Care Med.
10:
38-47,
1994.
15.
Denburg, J. A.,
J. Gauldie,
J. Dolovich,
T. Ohtoshi,
G. Cox,
and
M. Jordana.
Structural cell-derived cytokines in allergic inflammation.
Int. Arch. Allergy Appl. Immunol.
94:
127-132,
1991[Medline].
16.
DeWet, J. R.,
K. V. Wood,
M. DeLuca,
D. R. Helinski,
and
S. Subramani.
Firefly luciferase gene: structure and expression in mammalian cells.
Mol. Cell. Biol.
7:
725-737,
1987[Medline].
17.
Emmel, E. A.,
C. L. Verweij,
D. B. Durand,
K. M. Higgins,
E. Lacy,
and
G. R. Crabtree.
Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation.
Science
246:
1617-1620,
1989[Medline].
18.
Giffin, W.,
H. Torrance,
D. J. Rodda,
G. G. Prefontaine,
L. Pope,
and
R. J. Hache.
Sequence-specific DNA binding by Ku autoantigen and its effects on transcription.
Nature
380:
265-268,
1996[Medline].
19.
Kao, P. N.,
L. Chen,
G. Brock,
J. Ng,
J. Kenny,
A. J. Smith,
and
B. Corthesy.
Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90.
J. Biol. Chem.
269:
20691-20699,
1994
20.
Lees-Miller, S. P.
The DNA-dependent protein kinase, DNA-PK: 10 years and no ends in sight.
Biochem. Cell Biol.
74:
503-512,
1996[Medline].
21.
Maldonado, E.,
R. Shiekhattar,
M. Sheldon,
H. Cho,
R. Drapkin,
P. Rickert,
E. Lees,
C. W. Anderson,
S. Linn,
and
D. Reinberg.
A human RNA polymerase II complex associated with SRB and DNA-repair proteins.
Nature
381:
86-89,
1996[Medline].
22.
Matsumoto-Taniura, N.,
F. Pirollet,
R. Monroe,
L. Gerace,
and
J. M. Westendorf.
Identification of novel M phase phosphoproteins by expression cloning.
Mol. Biol. Cell
7:
1455-1469,
1996[Abstract].
23.
Minakuchi, R.,
M. C. Wacholtz,
L. S. Davis,
and
P. E. Lipsky.
Delineation of the mechanism of inhibition of human T cell activation by PGE2.
J. Immunol.
145:
2616-2625,
1990
24.
Mouzaki, A.,
and
D. Rungger.
Properties of transcription factors regulating interleukin-2 gene transcription through the NFAT binding site in untreated or drug-treated naive and memory T-helper cells.
Blood
84:
2612-2621,
1994
25.
Mouzaki, A.,
R. Weil,
L. Muster,
and
D. Rungger.
Silencing and trans-activation of the mouse IL-2 gene in Xenopus oocytes by proteins from resting and mitogen-induced primary T-lymphocytes.
EMBO J.
10:
1399-1406,
1991[Abstract].
26.
Nair, A. P.,
S. Hahn,
R. Banholzer,
H. H. Hirsch,
and
C. Moroni.
Cyclosporin A inhibits growth of autocrine tumour cell lines by destabilizing interleukin-3 mRNA.
Nature
369:
239-242,
1994[Medline].
27.
Rao, A.
NFATp: a transcription factor required for the co-ordinate induction of several cytokine genes.
Immunol. Today
15:
274-281,
1994[Medline].
28.
Serfling, E.,
A. Avots,
and
M. Neumann.
The architecture of the interleukin-2 promoter: a reflection of T-lymphocyte activation.
Biochim. Biophys. Acta
1263:
181-200,
1995[Medline].
29.
Shaw, J. P.,
P. J. Utz,
D. B. Durand,
J. J. Toole,
E. A. Emmel,
and
G. R. Crabtree.
Identification of a putative regulator of early T cell activation genes.
Science
241:
202-205,
1988[Medline].
30.
Shelhamer, J. H.,
S. J. Levine,
T. Wu,
D. B. Jacoby,
M. A. Kaliner,
and
S. I. Rennard.
NIH conference. Airway inflammation.
Ann. Intern. Med.
123:
288-304,
1995
31.
Ting, N. S.,
P. N. Kao,
D. W. Chan,
L. G. Lintott,
and
S. P. Lees-Miller.
DNA-dependent protein kinase interacts with antigen receptor response element binding proteins NF90 and NF45.
J. Biol. Chem.
273:
2136-2145,
1998
32.
Uetsuki, T.,
A. Naito,
S. Nagata,
and
Y. Kaziro.
Isolation and characterization of the human chromosomal gene for polypeptide chain elongation factor-1 alpha.
J. Biol. Chem.
264:
5791-5798,
1989
33.
Venkataraman, L.,
D. A. Francis,
Z. Wang,
J. Liu,
T. L. Rothstein,
and
R. Sen.
Cyclosporin-A sensitive induction of NFAT in murine B cells.
Immunity
1:
189-196,
1994[Medline].
34.
Wang, J.,
C. H. Chou,
J. Blankson,
M. Satoh,
M. W. Knuth,
R. A. Eisenberg,
D. S. Pisetsky,
and
W. H. Reeves.
Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens.
Mol. Biol. Rep.
18:
15-28,
1993[Medline].