Glucocorticoid hormones downregulate histidine decarboxylase
mRNA and enzyme activity in rat lung
Cynthia A.
Zahnow1,
Pertti
Panula2,3,
Atsushi
Yamatodani4, and
David E.
Millhorn1,5
1 Department of
Physiology, University of North Carolina, Chapel Hill, North
Carolina 27599; 2 Department of
Biology, Åbo Akademi University, Biocity, Åbo
FIN-20520; 3 Division of Anatomy, Department of
Biomedical Sciences, University of Helsinki, Helsinki, Finland 00170;
4 Department of Medical Physics,
Osaka University, Osaka 565, Japan; and
5 Department of Molecular and
Cellular Physiology, University of Cincinnati, Cincinnati, Ohio
45267
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ABSTRACT |
Histidine decarboxylase (HDC) is the primary
enzyme regulating histamine biosynthesis. Histamine contributes to the
pathogenesis of chronic inflammatory disorders such as asthma. Because
glucocorticoids are effective in the treatment of asthma, we examined
the effects of 6 h of exogenously administered dexamethasone
(0.5-3,000 µg/kg ip), corticosterone (0.2-200 mg/kg ip), or
endogenously elevated corticosterone (via exposure of rats to 10%
oxygen) on HDC expression in the rat lung. HDC transcripts were
decreased ~73% with dexamethasone treatment, 57% with
corticosterone treatment, and 50% with exposure to 10% oxygen.
Likewise, HDC enzyme activity was decreased 80% by treatment with
dexamethasone and corticosterone and 60% by exposure to 10% oxygen.
Adrenalectomy prevented the decreases in HDC mRNA and enzyme activity
observed in rats exposed to 10% oxygen, suggesting that the adrenal
gland is necessary for the mediation of hypoxic effects on HDC gene
expression. These results demonstrate that corticosteroids initiate a
process that leads to the decrease of HDC mRNA levels and enzyme
activity in rat lung.
hypoxia; asthma; histidine decarboxylase gene; histamine; corticosterone
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INTRODUCTION |
HISTAMINE IS PRODUCED by the decarboxylation of
L-histidine via the pyridoxal-requiring enzyme
histidine decarboxylase (HDC). In eukaryotes, HDC is the primary enzyme
responsible for synthesizing histamine and is consequently a crucial
regulatory step for histamine biosynthesis. Histamine contributes to
the pathogenesis of obstructive lung diseases such as asthma primarily
through its actions on the inflammatory response, bronchial and
vascular smooth muscle, capillary and airway epithelial permeability,
and the secretion of mucus and serous fluid (6). Alveolar
hypoventilation leading to arterial hypoxemia and increased serum
levels of glucocorticoids are a common consequence of airway
constriction associated with asthma. Glucocorticoids are among the most
effective anti-inflammatory agents available for the treatment of
asthma. These steroids reduce the symptoms of asthma by
1) decreasing airway secretions via actions on mucous glands and vascular permeability,
2) decreasing airway inflammation
through inhibitions of inflammatory cell recruitment into the airways
and vasoconstriction of the vasculature,
3) decreasing airway reactivity, and
4) improving airway integrity and
thus function (22).
The anti-inflammatory actions of glucocorticoids are mediated through
glucocorticoid receptors (GRs), which are primarily localized to the
cytoplasm of target cells, but, upon binding of steroid hormone, the
activated complex moves to the nucleus where it dimerizes and binds to
the promoter region of DNA at consensus sites termed glucocorticoid
response elements (GREs) and either increases or decreases
transcription rates. Among the genes relevant to asthma,
glucocorticoids increase the transcription of lipocortin-1,
2-adrenoreceptor,
endonucleases, and secretory leukocyte inhibitory protein and inhibit
the transcription of many cytokine genes, inducible nitric oxide
synthase, inducible cyclooxygenase, inducible phospholipase
A2, endothelin-1, natural killer
cell receptors, and adhesion molecules (intercellular adhesion molecule-1; see Ref. 2). Despite extensive accumulated
knowledge about the anti-inflammatory actions of corticosteroids,
relatively little is known about the molecular mechanisms by which
these steroid hormones mediate their effects on the inflammatory
mediator histamine. We propose that glucocorticoids, endogenously
elevated during hypoxia associated with respiratory illness,
downregulate HDC gene expression. The resulting changes in availability
or storage of histamine may then contribute to the attenuation of the
inflammatory response. To address this question in rat lung, we have
studied the regulation of HDC mRNA, enzyme activity, and histamine
levels by glucocorticoids that were either exogenously administered or
endogenously elevated by exposure to 10% oxygen.
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MATERIALS AND METHODS |
Animals. Sprague-Dawley male rats were
housed in a controlled environment of 22°C and subjected to a
12:12-h dark-light cycle. They were fed pelleted Agway rat chow and
were given water ad libitum. At 30 days of age, the rats were
anesthetized via an intramuscular injection of a 5:1 mixture of
ketamine hydrochloride (Ketalar; 100 mg/ml)-xylazine (Rompun; 20 mg/ml)
and bilaterally adrenalectomized or sham operated. The rats were
postoperatively maintained with 0.85% saline for 2 wk. At 44 days of
age, the adrenalectomized, nonadrenalectomized (sham-operated), and
control (no surgery) rats were subjected to the various experimental
procedures and then killed by decapitation. To minimize any responses
that might have been caused by circadian changes in glucocorticoid levels, rats were killed between the hours of 1600 and 1800. Trunk blood and tissues were collected and frozen at
80°C for
later processing.
Steroid treatment. For the
dose-response studies, adrenalectomized, 44-day-old male rats were
intraperitoneally injected with a single bolus dose of either
0.5-3,000 µg/kg dexamethasone (Steraloids) or 0.2-200 mg/kg
corticosterone (Sigma). The steroid was first dissolved in 100%
ethanol (EtOH) and diluted to the appropriate dose using 1× PBS.
The final concentration of the vehicle was either 10% EtOH-90% PBS or
20% EtOH-80% PBS. Nonadrenalectomized (sham-operated) rats were
treated with vehicle alone. Six hours after treatment, rats were
killed, and tissues were removed and stored at
80°C for
later processing of HDC mRNA, HDC enzyme activity, and histamine
concentrations.
Hypoxia exposure. Forty-four-day-old
male rats were placed in a sealed Plexiglas chamber and exposed
continuously to humidified 10% oxygen for 6 h. Rats exposed to 6 h of
10% oxygen or room air were killed at ~1800, and those exposed for
shorter intervals were killed before 1800. The carbon dioxide produced
by the rats was vented through an opening in the chamber lid, and
oxygen levels were monitored within the chamber with an oxygen sensor
(Becton Dickinson Medical Systems). Rats were provided with food and
water ad libitum. Control (normoxic) rats were kept in an identical chamber, and all rats were subjected to a 12:12-h dark-light cycle. At
the end of the exposure to normoxia or hypoxia, rats were removed and
killed by decapitation.
Isolation of polyadenylated mRNA and Northern blot
analysis.
Poly(A)+-enriched RNA was isolated
and purified from lung and brain tissue by a modification of the
methods of Badley et al. (1) and Patel and Kurkinen (17). Tissue was
homogenized in a lysis buffer containing proteinase K (Boehringer
Mannheim), and mRNA was purified by affinity chromatography using
oligo(dT) cellulose (type 2; Collaborative Research). mRNA (10-20
µg) was resuspended in 10 µl of a glyoxal mixture (75 µl of DMSO,
30 µl of glyoxal, 15 µl of 0.2 M
Na2HPO4,
and 30 µl of H2O) and heated to
50°C for 1 h. Two and one-half microliters of 10× dye mix
(50% sucrose, 0.25% bromphenol blue, 1/10 vol, and 1×
Tris-borate-EDTA) were added to the samples, and the RNA
was size fractionated via electrophoresis in a 1% agarose gel prepared
in 10 mM
Na2HPO4
buffer. The RNA was then transferred in 20× sodium
chloride-sodium citrate buffer (SSC) to a Biotrans nylon
membrane. The membrane was prehybridized at 42°C for 2 h in a
solution containing 50% formamide, 4.8× SSC, 4.8×
Denhardt's solution, 48 mM
Na2HPO4,
1% glycine, 0.5% SDS, and 0.24 mg/ml salmon sperm DNA. Hybridization
proceeded at 42°C overnight in a solution containing 50%
formamide, 4.8% SSC, 1× Denhardt's solution, 20 mM
Na2HPO4,
0.1 mg/ml salmon sperm DNA, 0.5% SDS, 10% dextran sulfate, and 2 × 106
counts/min of radioactively labeled probe per milliliter
of hybridization solution. Rat HDC cDNA was labeled using
[
-32P]dCTP (3,000 Ci/mmol; Amersham) by nick translation (GIBCO BRL) as recommended by
the supplier.
-Actin oligonucleotide was end labeled using T4
polynucleotide kinase (Promega) and
[
-32P]ATP (New
England Nuclear).
To ensure that equivalent amounts of RNA were electrophoresed and
transferred, the level of
-actin mRNA in each lane was measured.
Densitometric measurements were made from the autoradiograms of the HDC
and
-actin hybridized Northern blots. Each measurement of HDC mRNA
was normalized to measurement of
-actin in the same sample and was
then expressed as a percentage of control or control mean
(±SE). Differences between groups were analyzed using the nonparametric, two-tailed Mann-Whitney statistical test.
Extraction and HPLC measurement of
histamine. Frozen tissues were sonicated in 10 volumes
of 2% perchloric acid for 15-20 s, at maximal setting, using a
sonifier cell disruptor (Heat Systems; Ultrasonics) and were
centrifuged at 4°C for 30 min at 12,000 rpm in an Eppendorf
microcentrifuge. The histamine content of the resultant supernatant was
measured as previously described using an HPLC fluorometric method
(25).
Extraction of HDC and assay for enzyme
activity. Frozen tissues were sonicated at maximum
setting in 8-10 volumes of an ice-cold solution (100 mM potassium
phosphate buffer, pH 6.8, 0.1 mM dithiothreitol, 0.01 mM pyridoxal
5'-phosphate, 1% polyethylene glycol-300, 20 µg/ml
phenylmethylsulfonyl fluoride, and 0.01 mM aminoguanidine) until
completely disrupted (23, 24) and centrifuged at 4°C for 30 min at
12,000 rpm in an Eppendorf microcentrifuge. The supernatant was
transferred into dialysis tubing (Spectra/Por MWCO 12-14,000) and
dialyzed three times against 100 volumes of the sonication solution at
4°C for ~20 h. Each extract was then divided into two 100-µl
portions for experimental and blank samples as well as a 10-µl
aliquot for Bradford protein assay (Bio-Rad).
-Globulin served as
the protein standard for generation of the standard curve for the
Bradford assay. One hundred microliters of L-histidine (1 mM) were added to serve as substrate for the enzyme activity assay, and
100 µl of water were added to the blank samples. The reaction
proceeded at 37°C for 4 h and was stopped by the addition of 20 µl of 20% perchloric acid. Samples were centrifuged at 12,000 rpm
for 15 min, and the histamine levels were measured by HPLC (25).
Corticosterone radioimmunoassay. Trunk
blood was collected from experimental rats into 50-ml polystyrene
tubes, allowed to clot on ice, and centrifuged to separate the serum.
Serum was removed and stored at
80°C until assayed. Total
serum corticosterone was measured using an Immuchem double antibody
(125I) radioimmunoassay kit from
ICN Biomedicals.
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RESULTS |
In rat lung, three transcripts of sizes 3.5, 2.6, and 1.6 kb were
detected using rat HDC cDNA as the probe (Fig.
1, A and B). Changes occurring in the levels
of the 3.5- and 2.6-kb transcripts were considered indicative of
changes in HDC mRNA, whereas the 1.6-kb transcript served as an
internal control since it remained unchanged throughout the
experiments. The levels of these two larger transcripts were decreased
in a dose-dependent manner in response to a 6-h treatment of
adrenalectomized rats with a bolus dose of dexamethasone
(0.5-3,000 µg/kg ip; Fig. 1, A
and C). These transcripts were
significantly decreased to below control values at 5.0 µg/kg
dexamethasone (P < 0.05), with a
maximum decrease of 73% observed at 2,000 µg/kg dexamethasone
(P < 0.05; Fig.
1C). Similarly, HDC enzyme activity
was decreased in a dose-dependent manner and was maximally reduced to
80% below that of the vehicle control value at 2,000 µg/kg
dexamethasone (P < 0.01; Fig.
2A). Unfortunately, changes in HDC protein levels could not be determined due to the unavailability of an antibody to HDC. No dose of
dexamethasone tested in these rats altered endogenous lung histamine
levels when compared with the vehicle control level (Fig.
2B).

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Fig. 1.
Dexamethasone mediates a dose-dependent decrease in histidine
decarboxylase (HDC) mRNA levels in rat lung.
A: representative Northern blot of HDC
poly(A)+ RNA purified from the
lungs of adrenalectomized rats injected with a single bolus dose of
dexamethasone (µg/kg ip) and killed 6 h later. Both vehicle
(adrenalectomized) and sham (nonadrenalectomized) rats were injected
with vehicle only [20% ethanol (EtOH)-80% PBS ip]. Three
HDC hybridizing transcripts of sizes 3.5, 2.6, and 1.6 kb were detected
using rat HDC cDNA as probe. B: to
control for loading and transfer errors, the membrane was stripped and
reprobed with oligonucleotides directed toward -actin.
C: densitometric measurement of each
HDC mRNA time point was normalized to measurement of -actin in the
same sample, expressed as %vehicle control and graphed. Each data
point is representative of an experimental group of
n = 4 rats except sham and 0.5 µg/kg
where n = 3 rats.
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Fig. 2.
Dexamethasone decreases HDC enzyme activity in a dose-response manner
(A) as determined by production of
histamine · µg 1 · h 1
but does not change endogenous histamine (HA) content
(B). Experimental conditions were as
described in Fig. 1. No. of rats for each experimental condition were
as follows: vehicle, n = 5; sham,
n = 5; 0.5, n = 7; 5.0, n = 6; 50, n = 7; 500, n = 6; 1,000, n = 6; 2,000, n = 5; 3,000, n = 5. * Significantly different from vehicle control (P < 0.01).
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All experiments in this study were conducted for 6 h because it was
determined that the maximal decrease in both HDC mRNA and enzyme
activity occurred after 6 h of treatment with a single bolus dose of
dexamethasone (3,000 µg/kg ip; Fig. 3,
A and
B). HDC mRNA levels were reduced to
71% below that of the vehicle control
(P < 0.05; Fig.
3B). Likewise, HDC enzyme activity
was reduced by 80% after 6 h, and this reduction was also observed at
12 and 24 h (P < 0.05; data not
shown). Histamine content did not significantly change from vehicle
control at any of the time points examined (data not shown).

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Fig. 3.
Maximal decrease in HDC mRNA occurs after 6 h of treatment with
dexamethasone. A: adrenalectomized
rats were treated with a single bolus dose of dexamethasone
(3,000 µg/kg ip) and killed at 2, 6, 12, and 24 h after injection.
Measurement of poly(A)+ mRNA was
normalized by division with the corresponding densitometric value
for -actin and expressed as percentage of vehicle control (10%
EtOH-80% PBS ip). Each time point contained
poly(A)+ mRNA prepared
from the lung of 4 separate rats.
B: 6-h Dex treatment
(3,000 µg/kg ip); 3.5- and 2.6-kb transcripts were reduced to
~71% below that of the vehicle control
(P < 0.05, n = 5 rats) * Significantly different from vehicle
control.
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To address the question of whether HDC gene expression is regulated by
both the synthetic glucocorticoid dexamethasone and the endogenous
glucocorticoid corticosterone, adrenalectomized rats were treated for 6 h with a single bolus dose of corticosterone (0.2, 2, 20, and 200 mg/kg
ip). Radioimmunoassay analysis of the serum from these rats
demonstrated that the administered corticosterone produced serum levels
that fall within the normal physiological range for rat corticosterone
levels (vehicle, 5.3 ± 1.6 ng/ml; 0.2 mg, 10.5 ± 5.4 ng/ml; 2 mg, 7.75 ± 5 ng/ml; and 20 mg, 124 ± ng/ml), and treatment with
the highest dose of corticosterone (200 mg/kg ip) resulted in a
pharmacological serum corticosterone concentration of ~1,839 ± 258 ng/ml. As was observed with dexamethasone, corticosterone
(0.2-200 mg/kg) caused a dose-dependent decrease in both
the 3.5- and 2.6-kb HDC transcripts, with a maximal decrease of 57%
below that of the vehicle control value
(P < 0.01) observed at the 200 mg/kg
dose (Fig. 4). Additionally, 20 and 200 mg/kg of corticosterone decreased HDC enzyme activity by ~80%
(P < 0.002), whereas histamine
levels remained unchanged compared with vehicle control (Fig.
5, A and
B). Thus similar changes in HDC gene
expression were obtained with exogenously administered dexamethasone
and corticosterone.

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Fig. 4.
Corticosterone mediates a dose-dependent decrease in HDC mRNA levels in
rat lung as determined by Northern blot analysis. Densitometric
analysis of HDC poly(A)+ RNA
purified from the lungs of adrenalectomized rats injected with a single
bolus dose of corticosterone (mg/kg ip) and killed 6 h later is shown.
Vehicle (Veh)-treated rats were injected with vehicle only (20%
EtOH-80% PBS ip). HDC hybridizing transcripts of sizes 3.5 and 2.6 kb
are indicated. Each measurement of HDC mRNA was normalized to
measurement of -actin in the same sample and expressed as %vehicle
control. No. of rats in each experimental treatment were as follows:
vehicle, n = 5; 0.2, n = 3; 2.0, n = 3; 20, n = 5; 200, n = 6. * Significantly different from vehicle control
(P < 0.01).
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Fig. 5.
Corticosterone decreases HDC enzyme activity in a dose-response manner
(A) as determined by
production of
histamine · µg 1 · h 1
but does not change endogenous histamine content
(B). Experimental conditions were as
described in Fig. 4. No. of rats for each experimental condition in the
HDC enzyme activity assay (HDC) and histamine determination (HA) were
as follows: vehicle, n = 7 (HDC and
HA); 0.2, n = 6 (HDC and HA); 2.0, n = 6 (HDC),
n = 5 (HA); 20, n = 10 (HDC),
n = 6 (HA); 200, n = 10 (HDC),
n = 6 (HA).
* Significantly different from vehicle control
(P < 0.002).
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The administration of high levels of dexamethasone and corticosterone
might reflect pharmacological rather than physiological serum levels of
the steroid in question. Therefore, in an effort to study the
regulation of HDC in a situation where glucocorticoid levels are
elevated, but more closely approximate physiological concentrations,
nonadrenalectomized rats were subjected to hypoxia, a general
physiological stress. Continuous exposure to 10% oxygen for 1 h
generated elevated levels of serum corticosterone of ~400 ng/ml,
which were four times that of the serum corticosteroid level for
normoxic rats. After 6 h of exposure, the serum steroid concentrations
were reduced to ~200 ng/ml, probably due to negative feedback by the
elevated corticosterone.
The rat's response to hypoxic exposure and the resultant changes in
the regulation of HDC gene expression in lung are complex. In an effort
to eliminate and thereby identify some of the hormonal factors that may
be involved in this response, the adrenal glands, the primary source of
corticosterone in the rat, were removed, and the rats were then
continuously exposed for 6 h to 10% oxygen. Both mRNA and enzyme
activity were decreased ~60 and 53%
(P < 0.005), respectively, in the
lungs of nonadrenalectomized, intact (sham) rats exposed to hypoxia for
6 h; however, adrenalectomized rats did not downregulate HDC mRNA or
enzyme activity (Figs. 6 and
7A).
These data indicate that the adrenal gland is necessary for the
mediation of hypoxic effects on HDC gene expression. Histamine levels
again remained unchanged in adrenalectomized and nonadrenalectomzed rats compared with the normoxic control value (Fig.
7B).

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Fig. 6.
Adrenalectomized (Adx) rats lose the ability to downregulate HDC mRNA
in response to hypoxic exposure as determined by Northern blot
analysis. Densitometric analysis of HDC
poly(A)+ RNA purified from the
lungs of adrenalectomized and nonadrenalectomized (intact or sham) rats
(n = 5) continuously exposed to 10%
oxygen (hypoxia) or room air (normoxia) for 6 h. HDC hybridizing
transcripts of sizes 3.5 and 2.6 kb are indicated. Each measurement of
HDC mRNA was normalized to measurement of -actin in the same sample
and expressed as %adrenalectomized normoxic control.
* Significantly different from adrenalectomized rats exposed to
normal air (P < 0.005).
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Fig. 7.
Adrenalectomy prevents the decreases observed in HDC enzyme activity
(A) when rats are exposed to
hypoxic conditions, but histamine levels remain unchanged
(B). Experimental conditions were as
described in Fig. 6. No. of rats for each experimental condition in the
HDC enzyme activity assay (HDC) and histamine determination (HA) were
as follows: Adx normoxia, n = 5 (HDC and HA); intact normoxia,
n = 6 (HDC),
n = 11 (HA); intact hypoxia,
n = 5 (HDC),
n = 4 (HA); Adx hypoxia,
n = 5 (HDC and HA). * Significantly
different from adrenalectomized rats exposed to normal air
(P < 0.005).
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DISCUSSION |
Multiple transcripts have previously been reported for HDC. HDC
transcripts of 3.5, 2.6, and 1.6 kb have been observed in rat brain (3)
and fetal rat liver (10); however, the latter does not contain the
1.6-kb transcript. Similarly, HDC transcripts of 3.7, 3.2, 2.9, and 1.6 kb have been detected in Drosophila head (5). Additionally, an HDC transcript of ~2.4 kb has been cloned
and sequenced from a human basophilic leukemia (KU-812-F) cell line
(26) and a human erythroleukemia cell line (28). There are very few
published studies that have addressed the basis for this transcript
heterogeneity. One such study investigated the functional analysis of
two HDC cDNA clones representing the 3.4- and 2.4-kb HDC transcripts of
a human basophilic cell line (KU-812-F). It was determined that the
3.4-kb mRNA is an alternately spliced transcript in which the 7th
intron (824 bp) is not spliced out and contains an in-frame translation
stop codon resulting in a truncated protein, whereas the 2.4-kb mRNA
was found to encode a functional HDC enzyme (14, 27). Likewise, it was
demonstrated in COS cells that the 2.6-kb HDC mRNA, isolated from rat
fetal liver, encodes a functional HDC (10). No published functional data exist for the smallest HDC transcript (1.6 kb). This mRNA could
code for a protein highly related to HDC and may therefore cross-react
with the HDC cDNA. Analysis of the 1.6-kb transcript using
oligonucleotides directed toward internal regions as well as the
5'- and 3'-ends has led us to speculate that the 1.6-kb transcript may be different from the published HDC cDNA sequences (data
not shown). Consequently, during our analysis of HDC mRNA, we have
focused on the changes occurring in the levels of the 3.5- and 2.6-kb
transcripts and have used the 1.6-kb transcript as an internal control
because its levels have remained unchanged throughout our experiments.
Our results from this study demonstrate that corticosteroids initiate a
process that leads to the decrease in HDC mRNA levels and enzyme
activity in rat lung. Steady-state levels of mRNA usually reflect a
balance between the rate of RNA transcription and RNA stability. We
have not determined which of these mechanisms might account for the
decrease in HDC mRNA, but we suspect that a reduction in transcription
rate may be involved. Sequence analysis of the rat HDC gene by our
laboratory has identified several putative regulatory elements,
including activator protein (AP)-1, AP-2, Oct 1, Sp1, CCAAT boxes,
hypoxia inducible factor (HIF)-1 binding sites, and five GRE-like
consensus sites (unpublished observations). There are several molecular
mechanisms that could account for a decrease in transcription rate. It
is well established that the GR interacts with c-Jun homodimers or
c-Jun-c-Fos heterodimers (AP-1) before DNA binding and results in an
inhibition of both of their abilities to activate transcription (15).
Additionally, the nuclear ratio of Jun-to-Fos is important for
determining whether interactions with the GR will have a negative or
positive effect on transcription. Another possible mechanism is that
GREs on the HDC gene function as a composite response element in which
the GR not only binds to DNA but also interacts with other
trans-acting factors to inhibit
transcriptional activity. Jun/Fos and HIF are likely candidates because
AP-1-like binding sites and HIF-1 binding sites have been
tentatively identified near several GRE-like sequences within the HDC
gene (unpublished observations).
Regulation of HDC gene expression by glucocorticoids may be occurring
via direct or multiple indirect pathways. It is well established that
GRs are present in rat lung (4). It is possible that the GR may
activate a different set of genes or a second messenger pathway that in
turn regulates HDC and can be additionally activated by something other
than GR.
In experimentation with intact animals, it is difficult to isolate the
variable under investigation from other physiological factors that may
also affect the condition being evaluated. Our data
support the hypothesis that glucocorticoids, in part, mediate the
hypoxic downregulation of HDC mRNA because the regulatory response is
blocked in adrenalectomized animals. However, we cannot discount the
possibility that factors other than glucocorticoids may be influencing
the regulation of HDC gene expression. For example, hypoxic stress is
associated with elevated serum levels of corticosterone, ACTH, and
catecholamines (18, 21). ACTH levels were not measured in our study but
are reported to be elevated during hypoxia (18, 21) and downregulated
(via negative feedback) as doses of exogenously administered steroids
are increased. If so, it seems unlikely that these opposite
fluctuations in the levels of ACTH could account for the consistent
downregulation of HDC mRNA and enzyme activity during both experimental
paradigms. The adrenal gland is the sole source of glucocorticoids and
mineralcorticoids and also produces androgens, estrogens, progestins,
and catecholamines. This gland is necessary for mediation of the
regulatory effects of hypoxia because adrenalectomized rats lose the
ability to downregulate HDC gene expression in response to hypoxic
exposure (Figs. 6 and 7, A and
B). Studies have demonstrated that
removal of the adrenal gland can exacerbate the inflammatory response,
suggesting that endogenous steroids can suppress inflammation (22). It
is possible, however, that other adrenal steroids or catecholamines
might be involved in the regulation of HDC gene expression.
Interestingly, endogenous histamine concentrations within the rat lung
did not change significantly during any of the experimental conditions
used in this study. Several explanations may account for this
observation. In the lung, the majority of histamine is stored within
mast cell granules and exhibits a very slow turnover rate (13). The
80% decrease observed in HDC enzyme activity may not produce an equal
decrease in histamine stores over a 6-h period. In order for the amine
to be metabolized, it must be released from its storage granules.
Degranulation of mast cells is a complex process, and, although hypoxia
has been shown to induce histamine release (7), it is not known whether
the experimental paradigms used in this study caused degranulation.
Corticosteroids do not directly inhibit histamine release from lung
mast cells; however, steroid treatment is associated with a decrease in
mucosal mast cell numbers (2). If a decrease in mast cell number was
responsible for the observed decreases in HDC mRNA and enzyme activity,
a decrease in histamine levels might also be expected to occur. Additionally, the activity of histamine-metabolizing enzymes may also
be downregulated by glucocorticoids. We speculate that, over longer
periods of time, the reduced levels of HDC mRNA and enzyme activity
will result in a decreased production and consequently a decreased
tissue level of histamine.
In agreement with our results, several studies in rat and mouse lung
have shown that glucocorticoid hormones may be involved in the
reduction of HDC enzyme activity (8, 19, 20). In contrast to our
studies in rat lung, glucocorticoids have been shown to activate HDC
gene expression in mastocytoma and basophilic leukemia cell lines.
Experiments with cell lines provide valuable information but must be
interpreted with caution because they do not precisely duplicate the
conditions and multiple variables that exist in the whole animal.
Dexamethasone increased both histamine content and the de novo
synthesis of HDC from cultured mouse mastocytoma P815 cells and
cultures of fetal rat hepatic cells (9, 16). This regulation was
further studied by examining the synergistic effects of glucocorticoids
and the protein kinase C activator 12-O-tetradecanoylphorbol
13-acetate (TPA) on HDC synthesis from mouse P815 cells (11).
Previously, it had been shown that activation of protein kinase C by
phorbol 12-myristate 13-acetate or oleoyl acetylglycerol led to
increased synthesis of HDC in rat basophilic leukemia cells, RBL-2H3
cells, and human basophilic leukemia cells, KU-812-F (12, 14). In
accordance, the TPA studies demonstrated that protein kinase C may be
involved in the glucocorticoid-induced synthesis of HDC in mouse
mastocytoma cells (11). Taken together, these results suggest that the
effects of glucorticoid hormones on HDC gene expression may be tissue
or cell specific.
In summary, glucocorticoid hormones do not appear to be responsible for
maintaining the constitutive expression of HDC in rat lung but do
appear to be involved in either the inactivation of HDC gene
transcription or reduction in HDC transcript stability. Additionally,
glucocorticoids reduce HDC enzyme activity, and it is not known whether
this reduction is due to a decrease in protein levels or the
posttranslational inactivation of enzyme activity. This downregulation
of HDC gene expression might, in part, account for the effectiveness of
glucocorticoids in the treatment of inflammatory diseases such as
asthma.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. Michael F. Goy, Pauline K. Lund, Kenneth
Korach, Philip A. Bromberg, and John H. Schwab for guidance and
critical reading of this work. We also thank Luisa E. Brighton for
assistance with the animal studies and Dr. Edward E. Lawson for
generous support.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-34919 (E. E. Lawson), HL-33831, and HL-59945 (D. E. Millhorn).
Address for reprint requests: D. E. Millhorn, Dept. of Mol. and Cell
Physiology, Univ. of Cincinnati, PO Box 670576, Cincinnati, OH
45267-0576.
Received 13 May 1997; accepted in final form 14 April 1998.
 |
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