The Interleukin-1 Type 2 Receptor Gene Displays Immediate
Early Gene Responsiveness in Glucocorticoid-stimulated Human Epidermal
Keratinocytes*
Walter J.
Lukiw
,
Jorge
Martinez¶,
Ricardo Palacios
Pelaez¶, and
Nicolas G.
Bazan
§
From the
Louisiana State University Medical Center,
Neuroscience Center and Department of Ophthalmology, New Orleans,
Louisiana 70112-2272 and ¶ Research and Development Department,
IFIDESA-ARISTEGUI, Industrial Farmaceutica y De Especialidades SA,
Maria de Molina 37, Madrid 28006, Spain
 |
ABSTRACT |
Human epidermal keratinocytes (HEKs) in primary
culture (P2-P4) were used to study glucocorticoid
(GC)-mediated transcription of the genes encoding the constitutively
expressed interleukin-1 type 1 receptor (IL-1R1) and the inducible
interleukin-1 type 2 receptor (IL-1R2). Utilizing Northern dot blot
analysis and a quantitative reverse transcription-polymerase chain
reaction protocol for IL-1R1 and IL-1R2, dexamethasone and, in
particular, the budesonide epimer R were shown to effectively and
rapidly induce transcription from the IL-IR2 gene when compared with
IL-1R1 or
-actin RNA message levels in the same sample. Southern
blot analysis of newly generated IL-1R2 reverse
transcription-polymerase chain reaction products using end-labeled
IL-1R2 intron probes suggested that GC enhancement of IL-1R2 expression
was regulated primarily at the level of de novo
transcription. GC-induced IL-1R2 gene transcription displayed features
characteristic of a classical immediate early gene response, including
a signal transduction function, a relatively low basal abundance, a
rapid, transient induction, cycloheximide superinduction, actinomycin D
suppression, and a rapid decay of IL-1R2 RNA message. Parallel time
course kinetic analysis of IL-1R2 RNA message levels with Western
immunoblotting revealed tight coupling of de novo IL-IR2
gene transcription with translation of the IL-1R2 RNA message; a newly
synthesized (~46-kDa) IL-1R2 protein was detected in the HEK growth
medium as early as 1 h after budesonide epimer R treatment. These
data indicate that different GC compounds can variably up-regulate the
IL-1R2 response in HEKs through transcription-mediated mechanisms and, for the first time, suggest that a gene encoding a soluble cytokine receptor can respond like an immediate early gene.
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INTRODUCTION |
The immunosuppressive and anti-inflammatory activities of
glucocorticoids (GCs)1 are
not well understood, due largely to limited knowledge of their complex
activities at the level of gene expression. Among the cytokines that
mediate the cellular immune and inflammatory response, the
interleukin-1 (IL-1) signaling system plays a central role in diverse
cell types (1-3). The ~17-kDa IL-1 affects target cells initially
through two distinct types of transmembrane receptor complexes:
(a) an integral type 1 (~80-kDa) IL-1 receptor (IL-1R1) protein, and (b) an external type 2 (~68-kDa) IL-1
receptor (IL-1R2; Refs. 4-6) protein. IL-1R1 and IL-1R2 proteins,
which are both members of the immunoglobulin gene superfamily, each
share virtual identity in their extracellular IL-1 ligand binding
domain and transmembrane anchor; however, the IL-1R1 has a 213-amino
acid cytoplasmic domain essential for signal transduction, whereas the
IL-1R2 has a 29-amino acid cytoplasmic terminus incapable of signal
transfer (2-6). Signal transduction through the IL-1R1 can be relayed
via cytosolic IL-1R-associated kinase cascades, leading to I
B
phosphorylation and degradation, followed by NF-
B action on target
NF-
B-regulated gene promoters (7-9). These target genes can include
those coding for: (a) specific IL-1-converting enzymes (10),
(b) proteases that cleave the soluble extracellular domain
of IL-1R2 (11); (c) the de novo expression of
IL-1R1, IL-1R2, and related growth factor genes (2, 11, 12); and (d) transcriptional activator proteins such as AP1, NF-IL6,
and NF-
B, which, in turn, induce transcription from IL-1-sensitive, pro-inflammatory IEGs, such as cyclooxygenase-2 (13, 14). At least two
distinct ligand-mediated mechanisms can modulate autocrine, juxtacrine,
or paracrine IL-1 stimulation: (a) the cellular production
of the ~20-kDa IL-1 receptor antagonist, which competes with the
binding of secreted IL-1 to IL-1R1 at the cell surface (15), and
(b) the de novo expression of the
non-signal-transducing IL-1R2 "decoy" receptor which, through its
prominent cysteine-rich extracellular domain, acts as a local
membrane-bound scavenger of IL-1 (but not IL-1 receptor antagonist) or
serves as a soluble ~46-kDa molecular sink for IL-1 (4, 11) by virtue
of an extracellular protease cleavage site.
Here we have studied the effects of the GCs dexamethasone (DEX),
the novel nonhalogenated budesonide epimer R (BUDeR), and an equimolar
racemic mixture of budesonide S and R (BUDr; Fig. 1) on IL-1R1 and IL-1R2 RNA message and
receptor protein induction using human epidermal keratinocytes (HEK)
cells at passages P2-P4 in a primary culture test system.
HEK cells respond to a wide variety of cytokines and lipid mediators
(12, 14, 16) and provide a useful model to study IL-1 biology because
they both synthesize and secrete IL-1 and respond to it via intrinsic
IL-1R1- and IL-1R2-mediated pathways (17,
18).2 Northern dot blot
analysis, quantitative reverse transcription-polymerase chain reaction
(RT-PCR), and multiplex RT-PCR using human-specific IL-1R1, IL-1R2, and
-actin primer sets radiolabeled to high specific activity
(>109 dpm/µg) indicated that after only 1 h of HEK
cell stimulation, DEX and, in particular, BUDeR elicited a strong
induction of the non-signal-transducing IL-IR2 RNA message but not of
the IL-1R1 transcript. Southern blotting and RT-PCR unprocessed
transcript assay (19, 20) indicated that IL-1R2 RNA increases were
primarily the result of de novo IL-1R2 gene transcription.
Temporal analysis of IL-1R2 RNA message abundance with newly
synthesized IL-1R2 protein indicated that the induction of the IL-1R2
gene expression pathway was a relatively rapid event, fulfilling each
of the criteria for the classical cellular IEG response (21). A
~46-kDa IL-1R2 protein was detected by Western immunoblot analysis in
HEK whole cell extracts (WCXTs) and in 200-fold concentrated HEK
extracellular growth medium as early as 1 h, and increasing to 12+
h after BUDeR treatment. These results suggest that rapidly induced
IL1-R2 gene transcription, tight coupling to IL-1R2 message
translation, and secretion of the IL1-R2 protein can contribute to the
anti-inflammatory potential of GC compounds in HEKs by scavenging and
antagonizing extracellular cytokine IL-1 activity. Enhanced local
up-regulation of the IL-1R2 signaling pathway by nonhalogenated
16
,17
-substituted GCs such as BUDeR may make these compounds
particularly useful anti-inflammatory agents in epidermal keratinocytes
that play key structural and physiological roles in primary immune
defense and the inflammatory response in vivo.

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Fig. 1.
Comparative structures of DEX and the
non-9 -fluorinated, 16 ,17 -acetyl-substituted stereoisomeric
glucocorticosteroids BUDeR and budesonide epimer S. BUDr is an
equimolar racemic mixture of BUDeR and budesonide epimer S compounds.
Me, methyl; Pr, propyl.
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EXPERIMENTAL PROCEDURES |
Inducers, Antagonists, and Other Reagents--
DEX
(9
-fluoro16
-methyl-11
,17
,21-trihydroxy-1,4-pregnadiene-3,20-dione;
Sigma D-1756), BUDeR
(16
,17
-butylidenebis(oxy)-11,21-di-hydroxypregna-1,4-diene-3,20-dione), and BUDr (an equimolar racemic mixture of budesonide S and R; Fig. 1)
were solubilized as 5 µg/µl solutions in 99.9% dimethyl sulfoxide
(ACS reagent grade; Sigma D8779). Actinomycin D (Sigma A9415 dissolved
in dimethyl sulfoxide) and cycloheximide (Sigma C0934 dissolved in ACS
grade ethanol) were used at 1 and 10 µg/ml, respectively. 20 ml of
keratinocyte growth medium (KGM; Clonetics) were made (100 nM with respect to each GC) to treat HEK cells in T-75
(Corning 25110-75; 75 cm2) tissue culture flasks with these
compounds. The ligands DEX, BUDeR, and BUDr were solubilized into the
KGM by 5 min of vigorous vortexing. The KGM in which the cells were
incubated (37 °C in 5% CO2) was decanted and replaced
with ligand-containing KGM and then warmed to 37 °C over a time
course of 0-24 h. All other reagents, enzymes, and media were of the
highest grades commercially available and were used without further purification.
HEK Cells in Culture--
HEKs respond productively to a
wide variety of cytokines, lipid mediators, and GCs. Cryopreserved
normal HEK cells (obtained from pooled donors (Clonetics CC-2504;
HEK-Neo pooled) and received as frozen primary cultures) were grown to
80% confluence (~2-3 × 106 HEK cells/T-75 flask)
in 20 ml of KGM supplemented with a serum containing 7.5 mg/ml bovine
pituitary extract, 0.1 µg/ml human epidermal growth factor, 5 mg/ml
insulin, 50 µg/ml gentamycin/amphotericin, and 0.5 mg/ml
hydrocortisone in T-75 flasks at 37 °C in 5% CO2 according to the manufacturer's specifications (Clonetics). HEK cells
responded well to ligands between P2 and P4,
after which there was a graded decline in both the growth rate and
responsiveness to ligand induction. The HEK cell cultures used here,
which were obtained from pooled neonatal or adult donors, revealed
little difference in their individual responses to either DEX, BUDr, or
BUDeR stimulation, as measured by the induction of the IL-1R2 RNA
message (Figs. 3-6). For maximal ligand induction, cells were deprived
for 24 h to 0.5% of normal serum levels before the addition of GC
test compounds. 100 nM (final concentration) DEX, BUDeR, or
BUDr were added to HEKs, and cells were harvested at 0, 1, 3, 6, 12, and 24 h. For each treatment condition, HEKs were cultured in
duplicate T-75 flasks for the parallel isolation of either total RNA
for Northern blot and RT-PCR analysis or WCXTs for Western immunoblot detection.
Harvesting of HEK and Preparation of WCXTs--
All
extraction procedures were performed at 4 °C on wet ice. After each
incubation period with GC test compounds, the KGM was decanted and
replaced with 20 ml of Dulbecco's phosphate-buffered saline (Life
Technologies, Inc.) containing 1 mM phenylmethylsulfonyl fluoride (Sigma P-7626), 0.05 µg/µl aprotinin (Sigma A-6279), and
0.025 µg/µl leupeptin (Sigma L-2884). HEKs were scraped into a
suspension of phosphate-buffered saline containing these enzyme inhibitors and pelleted at 4 °C by centrifugation at 1400 × Gav for 10 min. HEK cellular pellets were gently
resuspended in 200 µl of a hypotonic buffer consisting of 20 mM HEPES (pKa = 7.55 at 20 °C), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, and 1% (v/v) aprotinin (which was made 0.5% (v/v) with SDS
(Sigma L-4509) just before use). HEK WCXTs were thoroughly resuspended
using a 200 µl Pipetman with a cut-off tip followed by repeated
pipetting on wet ice for 5 min. HEK WCXT protein concentrations were
determined using a dotMETRIC protein microassay (Chemicon, Temecula,
CA; dot sensitivity, 0.3 ng of protein) using bovine serum albumin
(bovine serum albumin radioimmunoassay grade; Sigma A-7888) as a standard.
Isolation of Total Keratinocyte RNA--
After each GC
induction time point, the KGM was completely replaced with 7.5 ml of
TRIzol reagent (Refs. 22 and 23; Life Technologies, Inc. catalog number
15596-026). Samples were then incubated for 5 min, with gentle shaking
at 25 °C to dissociate nucleoprotein complexes. After the addition
of 1.5 ml of reagent grade chloroform, suspensions were transferred to
sterile, diethylpyrocarbonate (Sigma D5758)-treated 15-ml conical
tubes, vigorously shaken for 5 min, incubated at 25 °C for 15 min,
and centrifuged at 5000 × Gav for 15 min
at 4 °C. The upper aqueous phase, containing total extracted RNA,
was transferred into sterile RNase-free 15-ml conical tubes, and 4 ml
of 100% (v/v) ACS reagent grade iyl alcohol were added. RNA was
precipitated overnight at
80 °C and then centrifuged at 9000 × Gav for 10 min at 0 °C. Supernatants were vacuum aspirated, and visible RNA pellets were washed twice with 10 ml
of 80% ACS reagent grade ethanol (v/v) and pelleted by centrifugation at 9000 × Gav for 10 min at 4 °C.
Supernatants were aspirated again, and the total RNA pellet was dried
under a vacuum for ~10 min over a bed of anhydrous calcium sulfate
(Drierite; W. A. Hammond). The partially dessicated RNA pellet was
then resuspended in RNase-free water to ~1 µg/µl by incubating
for 15 min at 60 °C in an Eppendorf 5436 thermomixer. Total RNA
concentrations were determined spectrophotometrically at
A260, and samples had
A260:A280 ratios of
2.1.
Northern Dot Blot Analysis--
Northern dot blots
containing up to 10 µg of alkaline-denatured total HEK RNA were
prepared using 9 × 12-cm Zeta-Probe GT Nylon membranes and a
Bio-Dot SF Microfiltration apparatus (Bio-Rad 170-6543). Membranes were
hybridized using ExpressHyb hybridization solution
(CLONTECH), and RT-PCR generated IL-1R1, IL-1R2, or
-actin cDNA probes (Table I)
end-radiolabeled by using [
-32P]dATP (~3000 Ci/mmol)
or by direct incorporation using [
-32P]dCTP (~3000
Ci/mmol; Amersham redivue). Membranes were washed twice for 30 min at
61 °C in 1 mM EDTA, 40 mM
NaHPO4, pH 7.2, and 5% SDS according to the
manufacturer's protocols.
RT-PCR and Multiplex RT-PCR Analysis--
Typically, 1.0 µg of
the total HEK RNA was reverse transcribed into cDNA using 200 units
of SuperScript II reverse transcriptase (Life Technologies, Inc.) in
the presence of 5 µM random hexamers, 500 µM each deoxynucleotide triphosphate, 20 units of RNasin
(Promega N2115), 50 mM Tris-HCl, 10 mM
dithiothreitol, 75 mM KCl, and 3 mM
MgCl2 in a 20-µl volume at 42 °C for 90 min. This
generated a "mini" cDNA library from total RNA isolated from
control and GC-treated HEK cells. After cDNA synthesis, samples
were treated with RNase H for 90 min at 37 °C to remove residual
template RNA. PCR used ~10% (v/v) of the first strand (reverse
transcriptase) reaction as a DNA template in the presence of 200 µM deoxynucleotide triphosphates, 2 mM
MgCl2, and 2 units of AmpliTaq polymerase (Perkin Elmer
Corp.) in a final volume of 50 µl. This reaction included either
separate (standard RT-PCR) or combinations of primer sets (multiplex
RT-PCR) of 100 pM of each forward and reverse human-specific primer for IL-1R1, IL-1R2, or
-actin (Stratagene catalog number 302010; Table I). Either unlabeled (for "cold" multiplex RT-PCR) or [
-32P]dATP (>5000 Ci/mmol;
Amersham redivue AA0018) 5'-end-labeled primers (for "hot"
multiplex RT-PCR) were used in these reactions. [
-32P]dATP isotopes were typically utilized >4 days
before their quality control reference date. Quantitative IL-1R1 and
IL-1R2 RT-PCR amplification (Fig. 2) was performed for 31 cycles after
5 min of denaturation at 94 °C, 1 min of annealing at 60 °C, 1 min of primer extension at 72 °C, and 7 min of final extension at
72 °C after the 31st cycle using a GeneAmp 9700 PCR System (Perkin Elmer Corp.). Typically, 15 µl of each cold multiplex PCR reaction were analyzed on a 1× TBE (89 mM Tris-borate and 1 mM EDTA, pH 8.3) 1.5% agarose gel in the presence of 0.5 µg/ml ethanol-recrystallized ethidium bromide (Life Technologies,
Inc.; 5585 UA). PCR products of the hot multiplex PCR were separated on
5% or 10% (acrylamide:bisacrylamide, 30:1) Mini Protean II ready gels
(Bio-Rad 161-1101 and 161-1109) in 0.5× TBE buffer. Gels were dried
onto Whatman No. 1 filter paper (Bio-Rad gel dryer model 583) for 90 min at 80 °C and quantitated via signal transfer onto PhosphorImager
storage screens.
Southern Analysis of Unspliced IL-1R2 Transcript
Levels--
Southern blotting and RT-PCR unprocessed transcript assay
were performed as described previously (19, 20) with the following modifications. Up to 5 µg of 0, 1, 3, and 6 h BUDeR-induced
total HEK RNA was fed directly into a One-Step RT-PCR Amplification System (Life Technologies, Inc., Superscript 10928-026) using either
IL-1R2- or
-actin-specific primers. 35 µl of this integrated RT-PCR reaction were resolved by electrophoresis through 1× TBE, 1.5%
agarose gels, followed by Southern transfer onto Zeta-Probe GT Nylon
membranes (Bio-Rad). Antisense oligonucleotides derived from human
IL-1R2 exon 7, IL-1R2 intron 7 and intron 8, or the
-actin coding
sequence (Table I) were 5'-end-labeled using
[
-32P]dATP (>5000 Ci/mmol; Amersham redivue AA0018)
and T4 polynucleotide kinase (Promega M4101), hybridized at 55 °C to
60 °C using ExpressHyb hybridization solution, and were washed
according to the manufacturer's instructions
(CLONTECH).
Western Analysis and Immunoblotting--
30 µg of control or
GC-treated HEK WCXTs or HEK KGM concentrated 200-fold using Dulbecco's
phosphate-buffered saline and Centricon-10 concentrators (Amicon,
Beverly, MA) were analyzed under reducing conditions on 10% acrylamide
Tris-glycine SDS gels (Bio-Rad Ready-Gels 161-0907); proteins were
transferred onto Hybond-P (Amersham RPN2020F) polyvinylidene difluoride
transfer membranes using a mini-Trans-Blot electrophoretic transfer
cell (Bio-Rad 170-3930). Membranes were blocked and probed with an
anti-human IL-1R2 rat monoclonal primary antibody (Cdw121b) that
exhibited no cross-reactivity with human IL-1R1 (Genzyme, Cambridge,
MA). Bound primary antibodies were detected with an anti-rat IgG
peroxidase-linked secondary antibody (Amersham NA932) and developed
with an ECL Plus Western blotting analysis system, according to the
manufacturer's instructions (Amersham RPN2132).
Data Analysis and Quantitation--
For cold multiplex-PCR,
agarose gels were stained in 0.5 µg/ml ethidium bromide, and images
were either photographed using Polaroid instant photography or
digitized using the Bio-Rad Gel Doc 1000 UV Gel Documentation System.
For hot multiplex-PCR, dried polyacrylamide gels were exposed to
phosphorimager storage screens, and signals were analyzed via a raster
scanning laser on a GS-250 molecular imager (Bio-Rad). To quantitate
Western analysis signals Coomassie Blue-stained gels were placed on a
UV/white light conversion screen (Bio-Rad 1707538) and photometrically
digitized using the GS-250 molecular imager. Relative intensities of
the IL-1R1 and IL-1R2 RNA signals were quantitated against the
-actin RNA signal in the same sample using phosphorimager analysis
and the data acquisition packages provided with each instrument. All
p values were derived from protected t tests or
least square means from a two-way factorial analysis of variance
(ANOVA).
IL-1R Promoter DNA and IL-1R2 RNA Sequence Analysis--
PCR
primers were designed using Hitachi Oligo DNA sequence analysis
software (Version 5.0). Human IL-1R1 and IL-1R2 gene promoter 5'
untranslated region and IL-1R2 mRNA sequence data were obtained through GenBankTM accession numbers L09701, U14177, U14178 and X59770,
respectively. RNA sequence analysis was performed using both
Kodak/IBI/Pustell (a modified Version 2.04) and Omiga 1.1.3 (Oxford
Molecular) RNA subsequence analysis packages.
 |
RESULTS |
Utilizing human-specific IL-1R1 and IL-1R2 cold or hot primer sets
(Table I) and purified IL-1R1 and IL-1R2 cDNA templates, it was
determined that at 31 cycles of PCR amplification, the masses of the
IL-1R1 and IL-1R2 input cDNAs were linear functions of both the
IL-1R1 and the IL-1R2 primary PCR products (Fig.
2). Similarly, quantitative
-actin PCR
analysis using human-specific
-actin primers at a 60 °C annealing
temperature has been described previously (23). To ascertain whether
the forward and reverse primer sets specific for human IL-1R1 and
IL-1R2 were compatible in the same PCR reaction conditions at a common
60 °C annealing temperature, pilot experiments were performed using
mixed IL-1R1 and IL-1R2 primer sets (Table I) after 3 h of either
DEX or BUDeR treatment of HEK cells. Results of a cold multiplex PCR
experiment using only these two primer sets are shown in Fig.
3A. The gel reveals that only
two bands in the GC-treated HEKs were generated at 340 and 574 bp,
corresponding to the expected size of the IL-1R1 and IL-1R2 primary PCR
products from published DNA sequences (GenBankTM accession numbers
L09701 and X59770, respectively). Control HEK samples (no DEX, BUDeR,
or BUDr) yielded a strong IL-1R1 (304-bp) signal but only a weak 574-bp
band, corresponding to basal levels of the IL-1R2 RNA message under
control (uninduced) conditions. In agreement with previous observations
(24), this finding demonstrates that human-specific IL-1R1 and IL-1R2
RNA messages could be both reliably and simultaneously quantitated in
single 0.5-ml PCR reaction tubes. Notably, simultaneous 3-h incubation
of combinations of DEX + BUDr or DEX + BUDeR in serum-deprived HEKs
showed no additive function in either IL-1R1 or IL-1R2 RNA message
induction (Fig. 3A).

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Fig. 2.
RT-PCR quantitation of IL-1R1 and IL-1R2 RNA
expression in HEK cells. At 31 cycles, the relationship of input
cDNA to IL-1R1 and IL-1R2 primary PCR product was in the linear
range of amplification.
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Fig. 3.
Quantitation of the IL-1R1 and IL-1R2 RNA
signal. A, cold (ethidium bromide-stained) RT-PCR utilizing
mixed primer sets for IL-1R1 and IL-1R2 in the same sample;
B, hot (radiolabeled) RT-PCR utilizing mixed
end-radiolabeled primer sets for IL-1R1, IL-1R2, and -actin in the
same sample. HEK cells were treated with DEX, BUDeR, and/or BUDr for
3 h. M1, 246-bp marker; M2, 200, 400, and
800-bp low DNA mass ladder marker (Life Technologies, Inc.).
B, -actin shows anomalous band spreading during multiplex
RT-PCR. Low intensity IL-1R2 RNA signals are detectable in
serum-deprived untreated HEK cells using either technique.
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Hot multiplex-PCR at 31 cycles may provide a more precise quantitation
of the levels of specific RNA messages within complex populations of
cDNA (as derived from the reverse transcription of total RNA)
because: (a) the fluorescence of ethidium bromide-stained cold gels is nonlinear with respect to varying DNA concentrations, making RT-PCR products difficult to quantify, and (b) only
5'-end radiolabeled primers labeled to high specific activity are
incorporated into the primary PCR product in direct proportion to the
amount of existing template in the original cDNA sample. Omission
of the reverse transcriptase mix yielded no PCR signals; similarly, RNase-free DNase treatment of the HEK RNA extracts yielded only the
expected primary PCR bands at 340 and 574 bp. The induction of the
IL-1R1, IL-1R2, and
-actin RNA messages with BUDeR, DEX, and BUDr in
HEK cells using this technique is shown in Fig. 3B. Data on
the abundance IL-1R1 and IL-1R2 RNA messages were normalized against
the RNA signal in the same sample for
-actin, a moderate-to-highly abundant DNA transcript (23, 25). IL-1R1 and IL-1R2 RNA message levels
in control ranged from less than ~1% to ~4% of signal for
-actin RNA (Fig. 4). The strongest GC
induction at 3 h of the IL-1R2 RNA over the IL-1R2 RNA control
signal was by BUDeR (9.2-fold; p < 0.02), followed by
DEX (6.6-fold, p > 0.04) and BUDr (5.2-fold, p > 0.06). For each of the GCs studied, the IL-1R1 RNA
signal level was slightly depressed at the 1 and 3 h time points
when compared with control levels at zero time (Figs. 3 and 4). Using either Northern dot blot analysis (Fig. 4A) or the hot
quantitative RT-PCR at 31 cycles for both IL-1R1 and IL-1R2 (Fig.
4B), time points of 0, 1, 3, 6, 12, and 24 h
established a time course over which DEX and BUDeR induced
transcription from the IL-1R2 gene in HEK cells. De novo
IL-1R2 gene transcription was almost completely abolished by
pretreating HEKs with the DNA transcriptional inhibitor actinomycin D
(Fig. 4, ACTD) at 1 µg/ml in KGM. Cycloheximide (Fig. 4,
CHX; 10 µg/ml) superinduced IL-1R2 but not IL-1R1 RNA message abundance (Fig. 4, A-C).

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Fig. 4.
Time course of induction of human IL-1R2
genes with DEX and BUDeR as monitored by Northern dot blot analysis
(A) and by quantitative RT-PCR using end radiolabeled
primers (B). Using either technique, IL-1R2 was found
to be maximally induced at 3 h. In these experiments, BUDeR was a
consistently stronger inducer of IL-1R2 gene transcription than DEX
(C). Actinomycin D (ACTD) strongly suppressed
DEX- or BUDeR-mediated induction of the IL-1R2 transcript.
Cycloheximide (CHX) consistently superinduced IL-1R2 but not
IL-1R1 RNA message. Exposure time for IL-1R1 and IL-1R2 or -actin
dot blots was 26 and 8 h, respectively; exposure time for IL-1R1,
IL-1R2, and -actin RT-PCR signals was 1 h. Significance over
IL-1R1 signal at zero time, dashed line; *,
p < 0.04; **, p < 0.01; ***,
p < 0.001 (ANOVA).
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PCR primers that lie in exons 4 and 8 of the IL-1R2 gene and from the
-actin coding region (Table I) were then used for the direct
amplification of 0, 1, 3, and 6 h BUDeR-induced HEK RNA using a
single-tube integrated RT-PCR. Samples were then subjected to agarose
gel electrophoresis, Southern blotting, and probing with an end-labeled
-actin oligonucleotide probe to demonstrate equivalent RNA message
levels and RT-PCR efficiencies, an end-labeled oligonucleotide from
exon 7 to measure processed IL-1R2 RNA levels, and end-labeled
oligonucleotides from introns 7 and 8, (separating exons 7 and 8 and
exons 8 and 9, respectively) of the IL-1R2 gene to measure the
abundance of unspliced IL-1R2 transcripts. To demonstrate the
quantitative nature of this assay for IL-1R2-amplified sequences, mixtures of total RNA from control and GC-treated HEKs were subjected to the same RT-PCR and Southern blotting conditions and IL-1R2 exon 7 hybridization protocols and were analyzed along with the other samples.
Quantitation of IL-1R2 signal demonstrates the linearity of this assay
over the range of IL-1R2 induction observed (Fig.
5, right panel). No unspliced
IL-1R2 transcript and little processed IL-1R2 RNA were detectable at
0 h; however both unprocessed (intron 7-probed) and processed
(exon 7-probed) IL-1R2 transcripts are detectable in HEK RNA at 1 and
3 h after GC induction. Similar results were obtained using an
IL-1R2 intron 8 probe (data not shown). This suggests that GCs are
inducing IL-1R2 RNA message, at least in part, by increasing productive
transcription from the IL-1R2 gene.

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Fig. 5.
BUDeR induces unprocessed IL-1R2 DNA
transcripts and processed IL-1R2 RNA message in HEK cells. HEK
cells were induced with BUDeR for 0, 1, 3, and 6 h; total RNA was
then isolated and subjected to RT-PCR using primers for both IL-1R2 and
-actin (Table I). Primary PCR products were then separated by
electrophoresis, Southern blotted, and hybridized to labeled
oligonucleotides derived from the -actin coding region, intron 7 of
the IL-1R2 gene, or exon 7 of the IL-1R2 gene. Membranes were
phosphorimaged, and exposure times were adjusted for comparison of each
probe. Lanes A-E contain total RT-PCR products from HEK
cells treated for 3 h with BUDeR and contain 100%, 75%, 50%,
25%, and 0% of the total HEK RNA. These samples were subjected to
electrophoresis, Southern transferred, and hybridized with a labeled
IL-1R2 exon 7 probe. The linearity of this assay using the standard
curve generated by samples A-E is shown in the bottom
panel.
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The parallel induction of the ~68-kDa IL-1R2 protein by DEX and
BUDeR is shown in Fig. 6. De
novo IL-1R2 protein synthesis was almost completely blocked with
cycloheximide when HEK cells were pretreated at 10 µg/ml in KGM. The
rapid appearance of both a ~68-kDa (cell-associated) and a ~46-kDa
(soluble) IL-1R2 protein in HEK WCXTs, suggesting rapid IL-1R2
extracellular cleavage, was closely associated with increasing IL-1R2
RNA message output at 3 h. This suggested a tightly regulated
transcription-to-translation mechanism operating in the GC-triggered
IL-1R2 signaling pathway. Induction coupling of the IL-1R2 RNA message
to translation into IL-1R2 protein is shown at the 3 h time point
in Fig. 7. Newly synthesized ~46-kDa
IL-1R2 protein was rapidly shed into the HEK pericellular medium and
was abundantly detected in both HEK WCXTs (Fig.
8A) and in 200-fold concentrated HEK KGM as early
as 1 h after GC induction (Fig. 8B).

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Fig. 6.
Induction of the human ~68-kDa
(membrane-associated) IL-1R2 protein with DEX and BUDeR in HEK cell
WCXTs, as detected by Western immunoblotting using a human
IL-1R2-specific monoclonal primary antibody, was significant at 3 h (p < 0.02). A ~46-kDa IL-1R2 (shed) protein
was also associated with HEK WCXTs at this time point. Cycloheximide
strongly blocked both DEX- and BUDeR-mediated induction of both
membrane-associated and soluble IL-1R2 protein. Significance over
IL-1R2 signal at zero time, dashed line; *,
p < 0.05; **, p < 0.02, ***,
p < 0.001 (ANOVA).
|
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Fig. 7.
Tight coupling of the induction of the IL-1R2
RNA message (top panel) with IL-1R2 protein (bottom
panel) in control, DEX-, BUDeR-, and BUDr-treated HEK cells
relative to the IL-1R1 control (dashed line) assayed at
3 h after GC induction is typical of an IEG response. *,
p < 0.04; **, p < 0.01; ***,
p > 0.05 (ANOVA).
|
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Fig. 8.
Fate of newly synthesized IL-1R2 protein.
A, Coomassie Blue-stained 10% TGSDS gel profile of HEK
WCXTs 0-24 h after BUDeR induction. B, Western blot of HEK
KGM concentrated 200-fold and probed with an anti-human IL-1R2 rat
monoclonal primary antibody (Genzyme). M, molecular weight
standards; numbers to the left of the gel are the molecular
weights of markers in A. Arrows at ~46 kDa
indicate the approximate migration of HEK-associated and shed IL-1R2
protein.
|
|
Lastly, the 1286-nucleotide IL-1R2 RNA message contains several
structural features resembling IEG transcription products, including
enrichment in adenine-uridine-rich elements, RNA instability elements
associated with rapidly degraded RNA messages (Refs. 26 and 27; Fig.
9). Primary cultures of HEK cells at
P2-P4 may provide one preferred test system for evaluating
GC-mediated IL-1, IL-1R1, and IL-1R2 gene signaling pathways because
these epidermal keratinocytes were found to be at least 10-fold more responsive to both DEX and BUDeR stimulation than HeLa ATCC CCL-2, a
transformed, IL-1-responsive human epithelioid cell line. No GC-mediated IL-1R2 genetic response was noted in WI38 (ATCC CCL-75) cells, a diploid human lung fibroblast cell line, when treated under
identical assay conditions (Fig.
10).

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Fig. 9.
Schematic diagram showing the positions
(arrows) of 17 adenine-uridine-rich RNA message instability
elements relative to the polyadenylation signal in the 1286-nucleotide
IL-1R2 RNA message (GenBankTM accession number X59770).
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Fig. 10.
Comparison of 3-h DEX- and BUDeR-mediated
induction of the IL-1R2 RNA message in HEK neonatal and adult primary
cell lines and in HeLa and WI-38 cells, relative to their respective
control levels (dashed line). HeLa and WI-38 cell
lines, transformed cells of IL-1-responsive epidermal and fibroblast
origin, respectively, showed statistically insignificant IL-1R2
induction (p > 0.45); primary cultures of either
neonatal or adult HEKs consistently displayed significant IL-1R2
induction by the GCs DEX or BUDeR. Significance over IL-1R2 signal at
zero time, dashed line; *, p < 0.04; **,
p < 0.01 (ANOVA).
|
|
 |
DISCUSSION |
DEX, BUDeR, GC Receptor Binding, and IL-1R Gene
Expression--
Despite a nonsymmetrical 16
,17
-acetyl
substitution and a lack of a 9
-fluoro atom in its steroid nucleus,
BUDeR has been shown to have an 11-fold higher binding affinity for the
GC receptor when compared with DEX (28). BUDeR also shows greater
potency than DEX in modulating NF-kB-DNA binding (14) and in
stimulating transcription from synthetic target genes regulated by
GC-responsive elements in their promoters (29). Stereospecificity of
the BUDeR 16
,17
-acetyl substitution at the GC receptor may also
be critical in GC-receptor complex activation because BUDeR was found
to induce IL-1R2 gene transcription 1.8-fold greater (p < 0.02) than an equivalent concentration of BUDr, a 1:1 racemic
mixture of the budesonide R and S epimers (Fig. 1). Notably, no
synergism was noted when combinations of the GCs (DEX + BUDr or DEX + BUDeR) were used in treating HEK cells (Fig. 3), suggesting the
recruitment of single DEX-, BUDr-, or BUDeR-mediated signaling pathways
during IL-1R2 gene induction via productive GC-GC receptor interaction (14, 30-36). BUDeR was found to be the most efficient in rapidly up-regulating IL-1R2 RNA message and IL-1R2 protein abundance; a small
effect was also noted on the depression of IL-1R1 RNA and protein
levels (Figs. 4 and 7). The high ratio of topical to systemic activity
of BUDeR when compared with DEX (28, 29), indicates that BUDeR may be
pharmacologically preferred when inhibiting IL-1 biology and IL-1R
expression in the treatment of inflammatory conditions associated with
epidermal keratinocytes and related cell types.
GCs and the Induction of IL-1R2 Gene Transcription--
GC
compounds can inhibit the activation of genes coding for cytokine
signaling such as IL-1 and cell surface receptors required in the
inflammatory response by limiting the availability of transcription factors to access their target promoters (31-33). A dramatic
inhibition of cis-acting transcription factor AP1-like-,
-interferon activation sequence-, and especially NF-
B-DNA binding
by the GCs DEX and BUDeR, temporally correlating to a reduction in
cyclooxygenase-2 message synthesis, has recently been demonstrated in
HEK cell lines (14). Specifically, AP1 and NF-
B are required for the activation of many cytokine and cytokine receptor genes (1, 8, 31).
GCs, via the GC-receptor complex, can directly interact with
transcription factor AP1 through AP1's N-terminal domain to moderate
gene activation (32, 33); similarly, GCs like DEX can inhibit NF-
B
activation by induction of the I
B-
gene and I
B-
inhibitory
protein, which ultimately sequesters the NF-
B regulatory element as
an inactive cytoplasmic complex (34, 35). However, GCs appear not to be
just broad spectrum pro-inflammatory-gene repressors, but rather
utilize pleiotropic strategies to potentiate anti-inflammatory
responses. These processes include the transcription-mediated up-regulation of several cell surface and soluble ligand receptors, such as the
2-adrenergic receptor (36) and the induction of the
IL-1R2 in human B and T lymphocytes (4), mononuclear phagocytes (6,
37), polymorphonuclear cells, IL-1R2-transfected fibroblasts (38) and
in epidermal cell lines (16-18).
Our results suggest that in HEKs at P2-P4, BUDr, DEX, and
especially BUDeR trigger an up-regulation of basal IL-1R2 gene
expression, because low levels of both processed IL-1R2 RNA and IL-1R2
protein were detected in control HEK total RNA and WCXTs at zero time (Ref. 39; Figs. 3-7). No significant induction of IL-1R1 RNA message was noted. The fact that the human IL-1R1 and IL-1R2 genes are encoded
by multiple, tandemly linked DNA elements located in an IL-1R
coding-rich region on chromosome 2q12-13 is noteworthy; however, each
IL-1R promoter appears to be under individual transcriptional control
(4, 40). The IL-1R1 and IL-1R2 gene promoters are encoded by three or
two different 5' exons, respectively, at this locus (40, 41), therefore
depending on the selection of variably spliced IL-1R gene 5' regulatory
regions, AP1- and/or NF-kB-DNA binding may be alternately utilized to
promote transcription from a specific IL-1R gene isotype. Notably, each
IL-1R2 promoter exon but not every IL-1R1 promoter exon contains a
GC-responsive element consensus sequence (40). These latter features
may allow greater flexibility in the IL-1R response to different
concentrations or combinations of extracellular signaling ligands.
The GC-induced IL-1R2 Gene Behaves Like an IEG--
Newly
generated IL-1R2 RNA message contains features characteristic of the
transcription products of IEGs (21). These include expression in
diverse cell types, a signal transduction function, relatively low
basal abundance, rapid transient induction after treatment with GCs,
cytokines, and mitogens such as phorbol 12-myristate 13-acetate (4, 24,
38), cycloheximide superinduction, actinomycin D repression, and rapid
decay of the IL-1R2 RNA message. Moreover, RNA sequence analysis of the
1286-nucleotide IL-1R2 RNA message (GenBankTM accession number X59770)
reveals 17 adenine-uridine-rich RNA instability elements typical of
cytokine, lymphokine, and proto-oncogene RNA messages that are
transiently expressed and rapidly degraded (21, 26, 27; Fig. 9).
Northern analysis, RT-PCR assay, unprocessed DNA transcript assay, the
rapid disappearance of IL-1R2 RNA signal after a 3-h GC stimulation of
HEKs with either DEX or BUDeR, and actinomycin D suppression of this
GC-induction suggest that the de novo GC-mediated IL-1R2
expression pathway is regulated primarily at the level of IL-1R2 gene
transcription, although post-transcriptional IL-1R2 RNA message
stabilization may provide auxiliary controls in other cell types (11,
16). Parallel time course kinetic analysis of newly synthesized RNA levels with Western immunoblotting also revealed a tight coupling of
de novo IL-IR2 gene transcription with translation of the
IL-1R2 RNA message; a newly synthesized (~46-kDa) IL-1R2 protein was detected in the HEK pericellular environment as early as 1 h after BUDeR induction.
In conclusion, the established function of IL-1R2 protein is to act as
a molecular trap to capture extracellular IL-1 and thereby compromise
the IL-1 signaling system (6, 11, 42, 43) as a strong negative
extracellular regulator of IL-1 action. Our evidence of the rapid
coupling of IL-1R2 gene transcription to IL-1R2 RNA translation into
protein, followed by the shedding of the ~46-kDa IL-1R2 into the
extracellular space, suggests that GC-triggered HEKs can deal rapidly
with local IL-1 levels via a classical IEG response. When compared with
DEX, nonfluorinated GCs bearing asymmetric 16
,17
-acetyl
substitutions such as BUDeR may elicit a stronger induction of the
IL-1R2 cytokine scavenger system. Such initiator elements of the IL-1R2
signal transduction pathway may therefore present future targets for
pharmacologic design in light of altered IL-1R2 gene expression in
pathological conditions of the gastrointestinal tract (44), in focal
cerebral ischemia (3, 45), in neuroinflammation (46), and in
neurodegenerative disorders of the brain (47, 48).
 |
ACKNOWLEDGEMENTS |
We thank Dr. Hilary Thompson
(Department of Ophthalmology, Louisiana State University Medical
Center) for statistical analysis of the IL-1R1 and IL-1R2 RNA and
protein signal data, Joelle Finley and Josephine Roussell (Louisiana
State University Medical Center Cell Culture Facility) for the
maintenance of human epidermal keratinocytes, and Dr. Harvey Herschman
(Molecular Biology Institute, University of California at Los Angeles)
for helpful suggestions and critical evaluation of the manuscript.
 |
FOOTNOTES |
*
This work was supported by a National Institutes of Health
EY02377 core grant and by a gift from the EENT Foundation. Part of this
research was presented in abstract form at the 6th International Conference on Platelet-activating Factor and Related Lipid Mediators held September 21-24, 1998 in New Orleans, Louisiana.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.
§
To whom correspondence should be addressed: Louisiana State
University Medical Center, Neuroscience Center and Dept. of
Ophthalmology, 2020 Gravier St., Suite D, New Orleans, LA 70112-2272. Tel.: 504-599-0831; Fax: 504-568-5801; E-mail: nbazan{at}lsumc.edu.
2
N. G. Bazan and W. J. Lukiw,
unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
GC, glucocorticoid;
HEK, human epidermal keratinocyte;
RT-PCR, reverse
transcription-polymerase chain reaction;
DEX, dexamethasone;
BUDeR, budesonide epimer R;
IEG, immediate early gene;
IL, interleukin;
IL-1R1, IL-1 type 1 receptor;
IL-1R2, IL-1 type 2 receptor;
NF-
B, nuclear factor
B;
AP1, activator protein 1;
BUDr, an equimolar
racemic mixture of budesonide S and R;
WCXT, whole cell extract;
KGM, keratinocyte growth medium;
ANOVA, analysis of variance;
bp, base pair(s).
 |
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