Prostaglandin-endoperoxide H Synthase-2 Expression in Human
Thyroid Epithelium
EVIDENCE FOR CONSTITUTIVE EXPRESSION IN VIVO AND IN
CULTURED KAT-50 CELLS*
Terry J.
Smith
§¶,
Timothy A.
Jennings
,
Daniela
Sciaky
, and
H. James
Cao
From the
Division of Molecular and Cellular Medicine,
Department of Medicine, the § Department of Biochemistry and
Molecular Biology, and the
Department of Pathology, Albany
Medical College and the Samuel S. Stratton Veterans Affairs Medical
Center, Albany, New York 12208
 |
ABSTRACT |
Prostaglandin-endoperoxide H synthase (PGHS) (EC
1.14.99.1) expression was examined in human thyroid tissue and in
KAT-50, a well differentiated human thyroid epithelial cell line.
PGHS-1 is found constitutively expressed in most healthy tissues,
whereas PGHS-2 is highly inducible and currently thought to be
expressed, with few exceptions, only in diseased tissues. Surprisingly,
PGHS-2 mRNA and protein were easily detected in normal thyroid
tissue. KAT-50 cells express high levels of constitutive PGHS-2
mRNA and protein under basal culture conditions. Compounds
usually associated with PGHS-2 induction, including interleukin-1
(IL-1
), phorbol 12-myristate 13-acetate, and serum transiently
down-regulated PGHS-2 expression. Human PGHS-2 promoter constructs
(
1840/+123 and
831/+123) fused to a luciferase reporter and
transfected into untreated KAT-50 cells exhibited substantial activity.
NS-398, a highly selective inhibitor of PGHS-2 could inhibit
substantial basal prostaglandin E2 production.
Exogenous IL-1 receptor antagonist or IL-1
neutralizing antibodies
could attenuate constitutive PGHS-2 expression in KAT-50 cells,
suggesting that endogenous IL-1
synthesis was driving PGHS-2
expression. Our findings suggest that normal thyroid epithelium
expresses high constitutive levels of PGHS-2 in situ and
in vitro and this enzyme is active in the generation of
prostaglandin E2. Thus, unprovoked PGHS-2 expression might
be considerably more widespread in healthy tissues than is currently believed.
 |
INTRODUCTION |
The human thyroid is a frequent site for the occurrence of
inflammatory and neoplastic disease. Much of the inflammation found in
this gland appears to be autoimmune, and the common processes include
Graves' disease and Hashimoto's thyroiditis (1). Thyroid immunity has
been the subject of substantial investigation because of the high
incidence of these autoimmune diseases in the general population.
Despite the particular susceptibility exhibited by thyroid to
inflammation, little insight currently exists into the molecular basis
for the peculiarities associated with thyroidal immunity. The currently
undefined role of arachidonate metabolites in the normal regulation of
thyroid growth and function, and in the mediation of thyroid disease,
is of potential importance.
Cyclooxygenases, also known as prostaglandin-endoperoxide H synthases
(PGHSs)1 (EC 1.14.99.1) are a
family of two bifunctional enzymes that catalyze the conversion of
arachidonate to prostaglandins (2). PGHSs are heavily glycosylated and
contain heme prosthetic groups. They are membrane-associated,
rate-limiting enzymes, each with two discrete, active sites catalyzing
both cyclooxygenase and peroxidase steps in the biosynthesis of
prostaglandins. Prostaglandin G2 is generated from
arachidonate, and then it is converted to prostaglandin H2.
The two PGHS isoforms are encoded by distinct human genes and localize
to different human chromosomes, and yet they have protein x-ray
crystallographic structures that are remarkably similar (3). PGHS-1 is
a constitutively expressed enzyme that is found at high levels of
abundance in most tissues and cells in culture (4-6). The levels of
PGHS-1 expression are relatively invariant with respect to tissue
involvement in disease or cytokine and mitogen action. It is the
enzymatic activity attributable to PGHS-1 that is currently thought to
contribute predominantly to basal PGE2 production in
healthy tissues and to maintain the integrity of renal and enteric
epithelium (2). In contrast, PGHS-2, the inflammatory cyclooxygenase,
is ordinarily not expressed in most tissues in states of health but can
be massively up-regulated by cytokines, growth factors, tumor
promoters, and serum in many cell types (7-15). PGHS-2 has been
demonstrated in situ in inflamed tissues (16). The pattern
of cellular distribution of PGHS-1 and -2 differs (17), suggesting that
the two enzymes might utilize discrete pools of arachidonate and
participate in different metabolic pathways. The question of why two
distinct but very similar enzymes might be co-expressed by the same
cell has not been answered, but PGHS-1 and -2 are thought to function,
at least in part, independently (2). PGE2 production
associated with the inflammatory response is currently believed to
emanate primarily from the activity of PGHS-2. Moreover, the decrease
observed in prostaglandin production in vivo and in
vitro following glucocorticoid treatment is attributable to the
down-regulation of PGHS-2 expression (9, 18).
Constitutive expression of PGHS-2 has been observed in a very few
tissues and cell types. For instance, in rat brain, the greatest
constitutive PGHS-2 expression is associated with the hippocampus, the
pyramidal cells of the piriform cortex, the amygdala, and neurons in
the neocortex (19, 20). The macula densa of the kidney was found to
express substantial levels of PGHS-2 under physiological conditions
(21). Unprovoked PGHS-2 has also been detected in bronchial epithelium
(22) and in granulosa (23), pancreatic islet (24), and hepatic stellate
cells (25). The function of PGHS-2 expressed in nonpathological states
is uncertain but opens the possibility that the biological role of this
cyclooxygenase isoform is not limited to the mediation of inflammatory
responses. Thus, the current categorization of cyclooxygenases as
representing a purely housekeeping enzyme (PGHS-1) on the one hand, and
a protein expressed exclusively in disease (PGHS-2) on the other, may
fail to embrace the apparent complexities of the prostanoid
biosynthetic machinery.
Very little is currently known about the expression of PGHS isoforms or
the production of prostanoids and other eicosanoids in the human
thyroid. The biosynthesis and action of lipid mediators in thyroid
tissue may be complex because multiple cell types normally reside or
are recruited there in states of disease. We have reported recently
that human thyroid fibroblasts can synthesize PGE2 and that
the production of this prostanoid is up-regulated by cytokines such as
IL-1
(26). Moreover, we have postulated that fibroblasts, by virtue
of their diverse array of small molecule expression, represent
potentially important orchestrators of the early events in tissue
remodeling (27). With regard to thyroid epithelial cells, recent
studies have suggested that the proliferation-promoting actions of
thyroid-stimulating immunoglobulins, as assessed in the FRTL-5 rat
thyrocyte line, are mediated through activation of phospholipase
A2 and arachidonate release and can be attenuated with
indomethacin, a nonselective cyclooxygenase inhibitor (28, 29).
Although the results of these studies imply that cyclooxgenase products
are involved in the growth-stimulating effects of thyroid-stimulating immunoglobulin on rat thyrocytes in vitro, the expression of
specific PGHS isoforms has not been examined previously.
In the present study, we assessed the expression of both PGHS-1 and -2 proteins by immunohistochemical means in situ in thin tissue
sections from a wide spectrum of thyroid disease. We report here the
unexpectedly high level of PGHS-2 protein expression in thyroid
follicular epithelium, both in normal tissue and in that involved in or
adjacent to disease. In addition, PGHS expression and activity were
assessed in an established, well differentiated human thyrocyte cell
line, KAT-50 (30). Surprisingly, KAT-50 cells express very high
constitutive levels of PGHS-2 mRNA and protein in culture and
produce PGE2. A substantial fraction of the
PGE2 generating activity in untreated KAT-50 cells can be attenuated with dexamethasone or NS-398, a highly selective inhibitor of PGHS-2 (31). Thus, we present compelling evidence that thyroid epithelium expresses PGHS-2 at high levels both in vivo and
in vitro and that this expression is constitutive and thus
may play an important role in normal thyroid function and immunity, as well as in the pathogenesis of disease.
 |
EXPERIMENTAL PROCEDURES |
Materials--
NS-398 as well as anti-PGHS-1 and PGHS-2
monoclonal antibodies were purchased from Cayman Chemical Co. (Ann
Arbor, MI). IL-1
was obtained from BIOSOURCE
(Camarillo, CA), and dexamethasone (1,4 pregnadien-9-fluoro-16
-methyl-11
,17
,21-triol-3,20-dione), phorbol 12-myristate 13-acetate (PMA), and cycloheximide were from
Sigma. Human PGHS-1 and PGHS-2 cDNA plasmids were gifts from Dr.
Donald Young (University of Rochester, Rochester, NY). Plasmids containing fragments of the human PGHS-2 promoter were generously provided by Dr. Stephen M. Prescott (University of Utah, Salt Lake
City, UT). Plasmid
1800pGL2 contained
1840/+123, and plasmid
800pGL2 contained the sequence
831/+123. pSV-
-galactosidase was
a gift of Dr. Brian Wilcox (Albany Medical College) and was used as a
transfection efficiency control. pGL2 Basic and pGL2 Promoter were
purchased from Promega (Madison, WI). Anti-IL-1
and IL-1
antibodies were from R&D Systems (Minneapolis, MN), and IL-1 receptor
antagonist (IL-1ra) was a gift from Amgen (Boulder, CO). Luciferase and
-galactosidase were measured with a Dual-Light kit from Tropix.
Human thyroid, abdominal adipose, and orbital connective tissues were
obtained from surgical waste. These activities have been approved by
the Institutional Review Board of the Albany Medical College.
Immunohistochemistry--
Thyroid tissue obtained from reactive
and neoplastic cases (37 total) was examined for the expression of
PGHS-2 protein. These cases included nodular hyperplasia (8 cases),
chronic thyroiditis (5 cases), diffuse hyperplasia of Graves' disease
(5 cases), follicular adenoma (8 cases), follicular carcinoma (3 cases), papillary carcinoma (3 cases), and medullary carcinoma (5 cases). In addition, at least one case from each of these categories
was also stained for PGHS-1. Five-µm-thick sections were cut from
formalin-fixed thyroid tissue that had been embedded in paraffin and
were applied to glass slides. After routine deparaffination and
rehydration, tissue sections were incubated in APK wash solution
(Ventana Medical Systems, Tuscon, AZ). Sections were placed in the
reaction chamber of a Ventana ES automated immunohistochemistry system,
which uses an indirect biotin-avidin detection method. Endogenous
peroxidase activity was blocked with 1% H2O2.
Sections were then incubated with primary anti-human PGHS-1 and PGHS-2
antibodies (Cayman) at a dilution of 1:100 for 24 min at 41 °C.
Following washes, sections were incubated with a biotinylated secondary
anti-mouse antibody, supplied by Ventana, for 8 min at 41 °C.
Isotype-matched antibodies were used as a negative control.
Cell Culture--
KAT-50 cells were a generous gift from Dr. K. Ain, University of Kentucky (Lexington, KY) (30). They were maintained
in a humidified, 5% CO2 incubator at 37 °C covered with
Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
bovine serum (FBS) and antibiotics. These cells have been characterized
as expressing, among other thyroid markers, thyroglobulin mRNA and the sodium/iodine symporter and were supplied to us in January 1997 at
passage 8 (30). Cells were passaged with gentle trypsin/EDTA treatment
and were utilized between the 10th and 35th passage. Medium was changed
every 3-4 days. Human orbital fibroblasts were obtained from explants
of surgical waste emanating from individuals with severe
thyroid-associated ophthalmopathy. Fibroblasts were cultivated as
described previously (32) in Eagle's medium supplemented with 10%
FBS, glutamine, and antibiotics. They were used between the 2nd and
12th passages.
Western Blot Analysis of PGHS Protein Expression--
Relative
levels of the cyclooxgenase proteins in thyroid and control tissues,
fibroblasts, and KAT-50 cells were determined by Western immunoblot
analysis utilizing monoclonal antibodies generated against human PGHS-1
and PGHS-2 and obtained from Cayman. Confluent cultures, usually
cultivated in 60-mm-diameter plates, were shifted to 1% FBS for
48 h. Monolayers were washed and harvested, and cellular protein
was solubilized in an ice-cold buffer containing 15 mM
CHAPS, 1 mM EDTA, 20 mM Tris-HCl (pH 7.5), 10 µg/ml soybean trypsin inhibitor and 10 µM
phenylmethylsulfonyl fluoride. Lysates were taken up in Laemmli buffer
and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and
the separated proteins were transferred to polyvinylidene difluoride
membrane (Bio-Rad). The primary antibodies (10 µg/ml) were incubated
with the membranes for 2 h at room temperature and the membranes
were then washed extensively and reincubated with secondary,
peroxidase-labeled antibodies for 2 h. Following washes, the ECL
(Amersham Pharmacia Biotech) detection system was used to generate the
specific signals. Resulting bands were quantitated densitometrically
with a BioImage (Milligen) scanner.
Northern Blot Analysis of PGHS-encoding mRNAs--
Total
cellular RNA was extracted using a method published by Chomczynski and
Sacchi (33) from thyroid and control tissues or from near-confluent
100-mm-diameter plastic plates of KAT-50 cells that had been incubated
without or with the test compounds indicated in the figure legends.
Following extensive rinsing with PBS, monolayers were covered with a
solution containing guanidium isothiocyanate (ULTRASPEC RNA isolation
systems, Biotecx Laboratories, Houston, TX), and RNA was precipitated
from the aqueous phase by addition of isopropanol, washed with 75%
ethanol, and solubilized in diethyl pyrocarbonate-treated water. Equal
amounts of RNA (usually 10-20 µg) were electrophoresed in 1%
agarose formaldehyde gels and transferred to Zetaprobe (Bio-Rad). The
integrity of the electrophoresed RNA was verified by UV inspection
following ethidium bromide staining. [32P]dCTP
random-primed (Bio-Rad) PGHS probes were hybridized in a buffer
containing 5× SSC, 5× Denhardt's solution, 50% formamide, 50 mM phosphate buffer (pH 7.2), 1% SDS, and 0.25 mg/ml
sheared, denatured salmon sperm DNA at 42 °C overnight. Membranes
were washed under high stringency conditions and exposed to X-OMAT AR film (Kodak, Rochester, NY) at
70 °C. To normalize the amounts of RNA transferred, either membranes were stripped according to the
manufacturer's instructions or the radioactivity was allowed to decay
and the RNA was rehybridized with a radiolabeled human GAPDH cDNA
probe. Radioactive DNA/RNA hybrids were quantitated by subjecting
autoradiographs to densitometric analysis.
For the PGHS-2 mRNA stability studies, 5,6-dichlorobenzimidazole
(DRB) (20 µg/ml), an inhibitor of gene transcription, was added to
the culture medium for the time intervals marked in Fig. 8, and the
abundance of steady-state PGHS-2 mRNA was quantitated with Northern
blot hybridization. The PGHS-2 mRNA signals were normalized to
their respective GAPDH signals. Data from two separate, identical
experiments were combined, and the normalized results were subjected to
a polynomial curve fit program (Delta Graph) using the least-squares
method to generate the curve shown in Fig. 8. To calculate the
t1/2 for PGHS-2 mRNA, the densitometric values
were modeled assuming first order decline employing the ADAPT II
program described by D'Argenio and Schumitzky (34). Those values that
were undetectable were coded as midway between the limit of detection
and zero in the analysis.
Transfection of PGHS-2 Promoter Plasmid Constructs into KAT-50
Cells--
KAT-50 cells were allowed to proliferate to a state of
60-80% confluence in 6-well plates covered with medium containing
10% FBS, and then monolayers were incubated with 1% FBS-enriched
medium for 24 h. Transfections were performed using Cellfectin
(Life Technologies) following the manufacturer's instructions.
Plasmids utilized for these studies included
1800pGL2, containing
1840/+123 and
800pGL2 containing
831/+123, and are thus 5 base
pairs upstream from the ATG of the human PGHS-2 promoter (35, 36).
These were fused upstream of the firefly luciferase reporter gene.
Control plasmid pGL2 was used to determine the basal level of
luciferase activity exhibited by these cells and pGL2 Promoter to
compare the strength of the PGHS-2 promoter to that of the SV40
promoter. pSV40-
-galactosidase was used as a transfection efficiency
control. 10 µl of Cellfectin was added to 100 µl of serum-free
medium without antibiotics, and the suspension was mixed gently with
the DNA, either the test plasmid (0.36 nM) or pGL2 (0.36 nM) together with pSV-
-galactosidase (0.4 µg).
Complexes were allowed to form over 15 min at room temperature before
being added to the washed monolayers. Cultures were incubated for
5 h at 37 °C in a 5% CO2 atmosphere, and then the
transfection mixture was removed and replaced with medium supplemented
with 1% FBS. Cultures were incubated for 24 h, cell monolayers
were washed in PBS, and 1× reporter lysis buffer (Tropix) was added
for 15 min at room temperature. The extract was vortexed and frozen at
80 °C. Samples were thawed and assessed for luciferase and
-galactosidase activity in a LUMI-VETTE luminometer (CHRONO-LOG).
The luciferase activities were corrected for their respective
-galactosidase levels and therefore reflect relative transfection efficiency.
PGE2 Assay--
PGE2 levels were
determined as described previously (14) utilizing a radioimmunoassay
(Amersham Pharmacia Biotech). Briefly, KAT-50 cells were inoculated in
24-well plates covered with medium supplemented with 10% FBS. One day
prior to experimental manipulation, culture wells were shifted to
medium with 1% FBS, and the following day, the test compounds
indicated in the legends to the figures were added. Thirty min prior to
monolayer harvest, medium was removed and replaced with 150 µl of PBS
with the respective additives. Following the incubation, the PBS was
removed quantitatively and subjected to the assay for PGE2
following the manufacturer's instructions. These studies were
conducted with three separate plates per treatment group. Data are
expressed as the mean ± S.E. of triplicate cultures from
representative experiments.
IL-1
and IL-1
Assays--
Details concerning these studies
have been published previously (15). Briefly, KAT-50 cells were allowed
to proliferate to near confluence in 24-well plastic culture plates
covered with medium containing 10% FBS. Monolayers were then shifted
to medium with 1% FBS for 16-24 h, and then test compounds were added
at the times indicated in the figure legends. Medium was removed, monolayers were washed with PBS, and cellular material was harvested in
a buffer containing 15 mM CHAPS, 1 mM EDTA, 20 mM Tris-HCl (pH 7.5), 10 µg/ml soybean trypsin inhibitor,
10 µM phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin,
and 0.5% Nonidet P-40. Cellular proteins (5 or 10 µg) were subjected
to specific ELISAs for IL-1
and IL-1
using kits from Immunotech
(Westbrook, ME). These assays were conducted according to the
manufacturer's instructions. Data are expressed as the mean ± S.E. of triplicate culture wells.
Statistics--
Data are usually expressed as the mean ± S.E. of replicate determinations unless indicated otherwise.
Statistical significance was determined by Student's
t-test.
 |
RESULTS |
PGHS-1 and PGHS-2 Protein Expression by Thyroid Follicular
Epithelium in Situ--
Sections of thyroid tissue, obtained from
surgical thyroidectomies performed as therapy for a wide variety of
diseases, were examined for PGHS protein expression by
immunohistochemical staining using isoform-discriminatory monoclonal
antibodies directed at either PGHS-1 or PGHS-2. As the photomicrographs
in Fig. 1 demonstrate, staining with
either PGHS-1 or PGHS-2 antibodies yielded very similar patterns. Both
enzymes are expressed abundantly in most follicular epithelial cells in
each of the cases examined. In addition, both antibodies labeled
vascular media, peripheral nerve, and some lymphoid cells. In contrast,
other stromal elements failed to stain with either antibody. The
epithelium exhibited diffuse, largely cytoplasmic immunoreactivity, in
lesional cells and normal elements adjacent to the lesions or in
separate blocks of nonlesional tissue. The intensity of staining was
generally moderate to strong and correlated with the cytoplasmic volume
rather than the presence or absence of disease. Little or no
nonspecific background staining was observed in sections stained with
isotype-matched controls (Fig. 1, middle left panel).

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Fig. 1.
Immunohistochemical examination
of thyroid sections for PGHS-1 and -2 proteins. Thyroid tissue
from a wide variety of disease processes was subjected to
immunohistochemical analysis using monoclonal antibodies directed at
isoform-specific epitopes. Top left panel, PGHS-2 expression
in Hashimoto's thyroiditis. Top right panel, PGHS-2
expression in papillary carcinoma of the thyroid. Middle left
panel, isotype control. Middle right panel, PGHS-1
expression in normal thyroid tissue. Bottom left panel,
PGHS-2 expression in normal thyroid, lower magnification. Bottom
right panel, PGHS-2 expression in normal thyroid, higher
magnification. Thyroid epithelial cells in normal and pathological
tissues express easily detectable levels of both cyclooxygenases. The
signal appears as a brown color. In contrast, other cell
types, including endothelial and interstitial cells appear to express
PGHS-2 only when in close proximity to inflammatory cells.
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We next determined whether PGHS-2 mRNA could be detected in thyroid
tissue. As the Northern blot in Fig.
2A indicates, a predominant 4.8-kb transcript is easily detected in total cellular RNA samples from
normal thyroid tissue, remote from any disease process. PGHS-1 mRNA
is also found in high abundance in these same samples as a single
5.2-kb band. Using Western blotting, PGHS-1 and -2 protein were also
detected in thyroid tissue from three different cases. (Fig.
2B). As the figure demonstrates, normal tissue from
multinodular goiter (patients 1 and 3) and Graves' disease (patient 2)
all express easily detectable levels of both isoenzymes. The levels of
the enzyme in the sample from the patient with Graves' disease are
somewhat lower than that found in the normal tissue from the two
multinodular glands. PGHS-1 appears as a 68-kDa protein band, whereas
PGHS-2 migrates to 72 kDa, consistent with our findings in human
fibroblasts (14). In contrast to the abundant PGHS-2 mRNA and
protein signals observed in thyroid tissue, normal intestinal fat
failed to express PGHS-2 mRNA on Northern analysis (Fig.
2C), and fat and normal striated muscle failed to exhibit
PGHS-2 protein on Western blots (Fig. 2D). Thus, it would
appear that both PGHS-1 and -2 mRNA and protein are abundantly
expressed in normal and pathological thyroid tissue, and this
expression localizes to epithelial cells.

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Fig. 2.
PGHS-1 and -2 mRNA and protein can be
detected by Northern and Western blot analysis, respectively, in human
thyroid tissue. A, thyroid tissues obtained from
surgical thyroidectomies were processed as described under
"Experimental Procedures," and cellular RNA was subjected to
electrophoresis, transferred to membranes, and analyzed. Northern blot
analysis was performed on 10 µg of cellular RNA using radiolabeled
probes generated from human PGHS-1 and PGHS-2 cDNAs. Patient
1, normal tissue remote from multinodular goiter; patient
2, normal tissue remote from a follicular neoplasm; patient
3, normal tissue remote from multinodular goiter. B,
thyroid tissue was homogenized, and the protein (30 µg per sample)
was subjected to PAGE and transferred to membranes as described under
"Experimental Procedures." The separated proteins were then
subjected to Western blot analysis using monoclonal antibodies directed
specifically against PGHS-1 and PGHS-2. Patient 1, normal
thyroid tissue from a multinodular goiter; patient 2, Graves' disease; patient 3, normal tissue from a
multinodular goiter. C, RNA was extracted from normal
intestinal fat and thyroid and (10 µg per sample) subjected to
Northern blot analysis of PGHS-2 and GAPDH mRNA levels.
D, protein (30 µg per sample) from normal intestinal fat,
normal striated muscle, and thyroid was electrophoresed and subjected
to Western blot analysis for PGHS-1 and PGHS-2 expression.
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|
Untreated KAT-50 Cells Express Constitutive PGHS-1 and PGHS-2
mRNA in Culture--
The recent reports of human thyroid
epithelial cell lines being isolated and characterized should
facilitate studies concerning thyroid growth and function. One of
these, KAT-50, derived from the non-neoplastic goiter of a young child,
expresses thyroglobulin and the sodium-iodine symporter (30). Moreover,
we have determined that these cells express high levels of thyrotropin
receptor mRNA.2 We
therefore determined whether the observations made concerning PGHS-2
expression in situ (Figs. 1 and 2) could be verified in these cells. Near-confluent cultures of KAT-50 cell monolayers were
analyzed by Northern blot analysis for the expression of PGHS-1 and
PGHS-2 mRNAs. Cultures were shifted to medium supplemented with 1%
FBS overnight prior to any experimental manipulations. As the blot
pictured in Fig. 3A indicates,
PGHS-1 mRNA is expressed at a high steady-state level by these
cells under basal culture conditions. The size of the PGHS-1 transcript
expressed by KAT-50 cells is 5.2 kb, and thus it resembles the mRNA
expressed in orbital and dermal fibroblasts (14), endothelial cells
(6), monocytes (37) and thyroid tissue (Fig. 2A). However,
it differs from the predominant 2.8-kb species that has been
demonstrated in some other human cell types (5). KAT-50 cells also
appear to express high levels of PGHS-2 mRNA under untreated
(basal) culture conditions. The PGHS-2 transcript appears as a single
predominant band of 4.8 kb, similar to that observed in several other
cell types (11-15) and in freshly obtained thyroid tissue. Because
this study was conducted by shifting the KAT-50 cultures to medium with
a reduced serum concentration (1% FBS), the readily detectable PGHS-2
mRNA levels were not a consequence of serum induction. Addition of dexamethasone (10 nM) to the culture medium for 6 h
resulted in a substantial down-regulation in the levels of PGHS-2
mRNA but had no effect on the abundance of the PGHS-1 transcript
(Fig. 3A). The attenuation of expression by dexamethasone
was transient in that levels of PGHS-2 mRNA had returned close to
control levels within 24 h of treatment. Glucocorticoids have been
shown previously to down-regulate PGHS-2 at the pretranslational level
in a wide variety of cell types that were activated with
proinflammatory molecules such as cytokines (9, 11).

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Fig. 3.
Northern blot analysis of steady-state PGHS-1
and -2 mRNA levels in untreated KAT-50 cells and those exposed to
IL-1 , dexamethasone, cycloheximide, and
PMA. A, KAT-50 cells were allowed to proliferate to
near confluence in 100-mm-diameter plates covered with medium
containing 10% FBS. They were then shifted to medium supplemented with
1% FBS for 24 h. Following incubation with the test compounds for
the treatment duration indicated in the figure, the monolayers were
harvested and processed for cellular RNA, which was subjected to
electrophoresis, transferred to membranes, and allowed to hybridize
with PGHS-1 and -2 cDNA probes. Following autoradiography,
membranes were stripped of radioactivity and rehybridized with a GAPDH
cDNA probe, and the signals were normalized. B,
near-confluent KAT-50 cells were shifted to medium containing either 1 or 10% FBS for 24 h. They were then treated with the compounds
indicated for 6 h, the monolayers were harvested, and RNA was
extracted and subjected to Northern analysis. The concentrations of the
compounds were as follows: IL-1 , 10 ng/ml; dexamethasone
(Dex), 10 nM; cycloheximide (cyclo),
10 µg/ml; PMA, 100 ng/ml). Dexamethasone and PMA could down-regulate
PGHS-2 mRNA at 6 h, but the effects of the compounds were
transient. The expression of PGHS-2 mRNA is at least partially
dependent on ongoing protein synthesis.
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In distinct contrast to the abundant PGHS-2 mRNA expressed in
untreated KAT-50 cells, orbital fibroblasts fail to express PGHS-2
constitutively, consistent with our earlier observations (14). When
these fibroblasts were treated with IL-1
(10 ng/ml) for 6 h,
they express high levels of PGHS-2 mRNA. Untreated orbital fibroblasts express PGHS-1 mRNA, which is invariant with regard to
IL-1
and dexamethasone treatment, in complete agreement with our
previous reports (14, 15). In contrast to its up-regulatory effects on
PGHS-2 in fibroblasts, IL-1
down-regulated levels of the transcript
in KAT-50 cells incubated in medium with 1% serum. Furthermore,
addition of the tumor promoter, PMA (100 ng/ml), a potent inducer of
PGHS-2 in many cultured cell types, also dramatically down-regulated
PGHS-2 mRNA levels to the limits of detection on Northern analysis
at 6 h (Fig. 3A). Similar to that of dexamethasone, the
effects of IL-1
and PMA were transient so that at 24 h, levels of the mRNA had returned to those observed in controls. We next examined whether the presence of 10% FBS in the medium would alter the
effects of IL-1
, PMA, and dexamethasone on PGHS-2 mRNA
expression. As the Northern blot in Fig. 3B suggests, the
higher serum concentration lowered PGHS-2 mRNA levels and addition
of any of these compounds failed to influence mRNA levels further.
To determine whether the high levels of PGHS-2 mRNA found to be
expressed by untreated KAT-50 cells were dependent on the synthesis of
an undefined protein, we assessed the impact that inhibition of ongoing
protein synthesis might have on cyclooxygenase expression. Treatment of
the cultures with cycloheximide (10 µg/ml), a concentration
associated with a greater than 90% inhibition of protein synthesis in
human cells (38), resulted in a rapid decrease in the levels of PGHS-2
mRNA (Fig. 3A). This is evident at both 2 and 4 h
after the inhibitor was added to the culture medium containing 1% FBS.
Thus, it would appear that ongoing protein synthesis is necessary for
the constitutive expression of PGHS-2 in KAT-50 cells. These results
are at marked variance with those in cells ordinarily not expressing
PGHS-2, such as fibroblasts and endothelial cells (11, 12). In earlier
studies, PGHS-2 mRNA was found to be rapidly and transiently
up-regulated by cycloheximide (11, 12), suggesting that the enzyme is
an early immediate response gene in these cells.
Untreated KAT-50 Cells Express Abundant PGHS-2 but Fail to Express
PGHS-1 Protein in Culture--
We next examined whether the high
levels of PGHS-1 and -2 mRNAs found in untreated KAT-50 cells
corresponded to expression of the respective proteins. Utilizing
monoclonal antibodies directed specifically against human PGHS-1 and
PGHS-2 in Western blots, we found that PGHS-2 protein was expressed in
untreated cultures (Fig. 4A).
PGHS-2 migrates as a 72-kDa protein (Fig. 4A). Fig. 4B demonstrates the very similar electrophoretic mobility
exhibited by PGHS-2 protein expressed by IL-1
-treated orbital
fibroblasts, untreated KAT-50 cells, and thyroid tissue. On the other
hand, Fig. 4A also demonstrates that PGHS-1 protein was
undetectable in the KAT-50 cells, whether untreated or following
incubation with IL-1
(10 ng/ml) for 16 h, despite the very high
levels of PGHS-1 mRNA expressed by these cells (Fig. 3). This
isoform can be easily detected in orbital fibroblasts as a 68-kDa band
on these blots and is invariant with regard to IL-1
treatment. The absence of PGHS-1 protein expression in KAT-50 cells in
vitro contrasts to the expression of this enzyme in
situ in thyroid tissue (Fig. 1). Thus, these cultured cells may
have escaped from factor(s) supporting PGHS-1 protein expression
observed in the intact gland. When IL-1
(10 ng/ml) was added to the
culture medium of nearly confluent KAT-50 cells, PGHS-2 protein
expression was rapidly and dramatically down-regulated (Fig.
5A). The levels were 11%
compared with basal values after 1 h of IL-1
exposure and had
begun to increase rapidly to 36 and 68% of control at 4 and 8 h,
respectively. Serum also appears to exert a substantial inhibitory
effect on PGHS-2 protein expression in KAT-50 cells (Fig.
5B), consistent with its effects on PGHS-2 mRNA levels. In a study in which FBS concentrations were varied and cultures were
exposed to serum for 24 h, the highest levels of PGHS-2 were observed in cells maintained in the absence of serum. Addition of 1%
serum lowered levels by 36%, and PGHS-2 expression was undetectable in
the presence of 10% FBS. This finding is in striking contrast to that
observed in fibroblasts, where serum induces strongly the expression of
PGHS-2 (11).

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Fig. 4.
Western blot analysis of PGHS-1 and -2 protein levels in KAT-50 cells and orbital fibroblasts.
A, near-confluent monolayers of KAT-50 cells and human
orbital fibroblasts, in this case from an individual with severe
thyroid-associated ophthalmopathy, were shifted to medium enriched with
1% FBS for 24 h. Sixteen h before monolayer harvest, some of the
cultures received IL-1 (10 ng/ml). Cellular protein was solubilized
as described under "Experimental Procedures," and 20 µg was
subjected to PAGE. The separated proteins were transferred to membrane
and Western blot analysis was performed using monoclonal antibodies
directed against human PGHS-1 and PGHS-2. Band intensities were
determined with a scanning densitometer. B, PGHS-2 protein
expression was compared in IL-1 (10 ng/ml)-treated orbital
fibroblasts, untreated KAT-50 cells, and normal thyroid tissue.
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Fig. 5.
IL-1 and serum
down-regulate PGHS-2 expression in KAT-50 cells. A,
near-confluent cultures of KAT-50 cells were shifted to medium
containing 1% FBS for 16 h. Nothing (control) or IL-1 (10 ng/ml) was added at time 0, and culture monolayers were harvested at
the times indicated along the abscissa. The cell proteins (30 µg per
sample) were subjected to PAGE as described under "Experimental
Procedures." Separated proteins were transferred, and Western blot
analysis performed using anti-PGHS-2 antibodies. Signals were generated
using the ECL system. B, near-confluent KAT-50 cell cultures
were shifted to medium containing graded concentrations of FBS as
indicated in the figure for 24 h. They were harvested and
processed as in A. Densities of resulting bands were
determined with a BioImage scanner.
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Constitutive PGHS-2 Expression in KAT-50 Cells Is Dependent upon
the Synthesis of IL-
--
We have begun to examine the factors
expressed by KAT-50 that might underlie the constitutive expression of
PGHS-2. Because IL-1
and IL-1
are actively synthesized widely in
many cell types and are important inducers of PGHS-2 (12, 13), we
determined whether endogenous IL-1 production could be related to
PGHS-2 expression in KAT-50 cells. We have found very recently that
IL-1
but not IL-1
expression and inducibility in human orbital
fibroblasts is a critical mediator of the up-regulation of PGHS-2 by
CD40/CD40 ligand engagement (15). To determine whether IL-1
and/or
IL-1
are expressed in KAT-50 cells, we performed specific ELISAs for these cytokines. As the data in Fig.
6A indicate, we could readily detect IL-1
under basal culture conditions. The level in cell layers
was 16.5 ± 1 pg/10 µg of protein (mean ± S.E.,
n = 3). When the cultures were treated with
dexamethasone (10 nM) under conditions that attenuate
PGHS-2 mRNA levels (Fig. 3), IL-1
was down-regulated to
undetectable levels. In contrast, IL-1
could not be detected in
untreated KAT-50 cells or those receiving dexamethasone (Fig.
6A). Thus, it would appear that the glucocorticoid might be
influencing the levels of PGHS-2 expression through actions on IL-1
synthesis. To test further the central role of IL-1
expression in
supporting constitutive PGHS-2, the effects of 10% FBS on the levels
of the cytokine were examined. This concentration of FBS elicited a
time-dependent and dramatic decrease in IL-1
levels so
that within 1 and 2 h of serum addition to the medium, the
cytokine concentration in KAT-50 cell-conditioned medium was reduced by
59 and 93%, respectively (Fig. 6A).

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Fig. 6.
KAT-50 cells express constitutive
IL-1 protein that can be down-regulated by
dexamethasone and serum. Neutralization of IL-1 down-regulates
constitutive PGHS-2 expression. A (left), cell
layers from a 24-well plate were allowed to proliferate to near
confluence and shifted to medium containing 1% FBS, and some were then
treated with dexamethasone (10 nM) for 16 h.
Monolayers were solubilized and subjected to an ELISA specific for
IL-1 and IL-1 . Data are expressed as the mean ± S.E. of
triplicate cultures from a representative experiment. A
(right), cell layers of KAT-50 cells were shifted from
medium containing 1% FBS to that with 10% serum for the times
indicated along the abscissa. The cells were then analyzed for IL-1
levels. B, Western analysis of PGHS-2 protein expression in
KAT-50 cells treated with nothing (control) or with neutralizing
antibodies against IL-1 (1 µg/ml), IL-1 (1 µg/ml), or with
Il-1ra (500 ng/ml) for 16 h. Cell layers were solubilized, 30 µg
of each sample was subjected to PAGE, and the separated proteins were
transferred to membranes and probed with monoclonal antibodies against
PGHS-2. The signals generated with ECL detection were scanned with a
BioImage densitometer. C, Northern analysis of PGHS-2
transcript levels in KAT-50 cells treated with IL-1ra (500 ng/ml) for
the duration indicated in the figure. Total cellular RNA (30 µg) was
electrophoresed, transferred, and hybridized with a PGHS-2 cDNA
probe and then with a GAPDH probe. Densities of the resulting bands
were determined, and the corrected PGHS-2 signals are shown.
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To determine directly whether the expression of IL-1
was proximately
related to the constitutive expression by KAT-50 cells of PGHS-2,
neutralizing antibodies against IL-1
(1 µg/ml), IL-1
(1 µg/ml), or exogenous IL-1ra (500 ng/ml) were added to the culture medium of near-confluent KAT-50 cultures. The cells were then assessed
for PGHS-2 protein expression by Western blot analysis. Anti-IL-1
antibody and IL-1ra, but not anti-IL-1
antibody, could substantially
down-regulate PGHS-2 protein levels when added to the culture medium
for 16 h (Fig. 6B). The inhibition with anti-IL-1
was 45%, whereas IL-1ra inhibited PGHS-2 protein expression by 97%
(Fig. 6B). As the Northern blot in Fig. 6C
indicates, addition of exogenous IL-1ra also dramatically attenuated
the PGHS-2 mRNA expression, suggesting that IL-1
is exerting a
pretranslational effect on PGHS-2 expression. These results are
consistent with endogenously generated IL-1
supporting the
constitutive expression of PGHS-2 in KAT-50 cells. In contrast, it
appears that endogenous IL-1
does not play an important role in the
basal expression of this cyclooxygenase. Moreover, it would appear that
the down-regulatory effects of both glucocorticoids and serum on PGHS-2
expression in KAT-50 cells are mediated, at least in part, through the
modulation of IL-1
synthesis.
PGHS-2 Promoter Activity Is Easily Detectable in Untreated KAT-50
Cells--
KAT-50 cells were transfected with a reporter gene to
assess the basal activity of the PGHS-2 promoter. The proximal
1840/+123 base pairs of the human PGHS-2 promoter fused to a
luciferase reporter were transfected into near-confluent cells. As the
data contained in Fig. 7 indicate, PGHS-2
promoter activity is easily detected in these cells under basal culture
conditions that was more than 62-fold higher than the activity of pGL2
Basic (control) plasmid. Moreover, the PGHS-2 promoter strength was
13-fold greater than that manifested by the SV40 promoter. A smaller
PGHS-2 promoter construct representing
831/+123 base pairs yielded
considerably less activity but was still approximately 35-fold above
the control plasmid. These results are entirely consistent with the
substantial PGHS-2 expression found in untreated KAT-50 cells and
provide insight into the extreme activity exhibited by this promoter. It is of considerable mechanistic relevance that both constructs demonstrating activity in the transfected KAT-50 cells contain several
regulatory elements including two putative NF-
B sites present in the
human PGHS-2 promoter. These are found at
214/
204 and
447/
437
nt (35).

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Fig. 7.
Untreated KAT-50 cells in culture exhibit
substantial PGHS-2 promoter activity. Near-confluent KAT-50 cells
were transfected with the 1840/+123 or 831/+123 PGHS-2 promoter
constructs fused to a luciferase reporter or the luciferase reporter
without (pGL2 Basic) or with the SV40 promoter (pGL2 Promoter) as
described under "Experimental Procedures." Cultures were
co-transfected with -galactosidase reporter plasmids, the activity
of which was assayed and used to correct each culture for transfection
efficiency. The data are expressed as the mean ± S.E. of
triplicate culture determinations from a single experiment that is
representative of three independent studies.
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Assessment of PGHS-2 mRNA Stability in Cultured KAT-50
Cells--
The 3' untranslated region of the human PGHS-2 mRNA
contains at least 22 AUUUA instability determinants (10, 36) and
therefore exhibits the potential for behaving as a rapidly turning over transcript. We therefore set out to determine whether the PGHS-2 mRNA was particularly long-lived in KAT-50 cells and whether this relative stability could underlie the high constitutive levels of
PGHS-2. The rate of mRNA turnover in KAT-50 cells was assessed by
blocking transcriptional activity. Cultures were treated with DRB (20 µg/ml), an inhibitor of gene transcription, for the intervals of time
indicated along the abscissa in Fig. 8.
As the data in the figure suggest, there is a slow decrease in the
steady-state levels of PGHS-2 mRNA over the first 2 h of DRB
addition to the culture medium. The transcript level decreases more
rapidly over the next 2 h so that PGHS-2 mRNA levels are
undetectable by 4-5 h. The calculated t1/2 of the
PGHS-2 mRNA in KAT-50 cells, based on the data from two independent
studies displayed in Fig. 8, is 3.22 h. Interestingly, the
degradation of PGHS-2 mRNA in these thyroid epithelial cells is
somewhat delayed compared with that observed in ECV304, an immortalized
human endothelial cell line, where the t1/2 was
approximately 1 h in DRB-treated cultures not exposed to any
cytokine (12). Unlike the studies we performed in the KAT-50 cells,
those earlier studies in ECV304 cells involved treating the cultures
initially with cycloheximide, and this inhibitor can influence mRNA
stability. Thus, although of interest, direct comparisons between the
studies in KAT-50 and ECV304 cells may be difficult.

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Fig. 8.
Assessment of PGHS-2 mRNA stability in
untreated KAT-50 cells. Near-confluent cultures of KAT-50 cells
were treated with DRB (20 µg/ml) for the times designated along the
abscissa, and total cellular RNA was extracted from monolayers. RNA was
subjected to Northern blot analysis by hybridizing 20 µg of cellular
RNA with a probe generated from the PGHS-2 cDNA as described under
"Experimental Procedures." Following autoradiography and
densitometric analysis, membranes were reprobed with a GAPDH probe, and
the data were normalized to the signal generated from the
standardization hybridization. The data derive from two independent,
identical studies, represented in the figure by different symbols. The
normalized data from the two studies were combined and subjected to a
polynomial linear regression best curve fit program (Delta Graph). The
calculated t1/2 for PGHS-2 mRNA is 3.22 h
as determined by the method of D'Argenio and Schumitzky (34).
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Untreated KAT-50 Cells Synthesize PGE2 with Activity
That Exhibits Sensitivity to PGHS-2 Selective Inhibition--
The
constitutive expression of PGHS-2 mRNA and protein in KAT-50 cells
implies that prostanoids such as PGE2 might be generated under basal culture conditions and be attributable to the activity of
this cyclooxygenase isoform. We therefore began to assess the production of PGE2 by quantitating the prostanoid released
from these cells into the culture medium. As Fig.
9A indicates, levels of
PGE2 under unstimulated culture conditions were low. When
graded concentrations of exogenous arachidonate (0-10
µM) were added to the culture medium, PGE2
production was increased substantially (Fig. 9A). Synthesis
of the prostanoid had nearly doubled (1.9-fold increase) with an
arachidonate concentration of 5 µM, and the PGE2 levels achieved with 10 µM were 5.6-fold
above those observed in cultures without exogenous arachidonate. The
nonselective compound, indomethacin, competitively inhibits both PGHS
isoforms (40). NS-398, in contrast, is a highly selective PGHS-2
inhibitor that exhibits a substantial preference for that isoform (31,
41). Addition of NS-398 (10 µM) to the medium of KAT-50
cells blocks PGE2 formation in arachidonate-treated KAT-50
cells by 57% (control, 1580 ± 195 pg/ml; NS-398, 680 ± 35, p < 0.002) (Fig. 9B). The inhibition
achieved was similar to that found in cultures receiving indomethacin
(10 µM), in which 77% of PGE2 production was
inhibited (360 ± 65 pg/ml, p < 0.001 versus control). Thus, a substantial fraction of
PGE2 produced by untreated KAT-50 cells can be attributed to the activity of PGHS-2. Dexamethasone (10 nM) could also
down-regulate the level of PGE2 synthesis (data not shown),
presumably reflecting the attenuation of PGHS-2 expression at the
pretranslational level.

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Fig. 9.
KAT cells in culture exhibit PGE2
synthetic activity under basal (untreated) culture conditions that can
be inhibited by indomethacin and NS-398. KAT-50 cells were
inoculated in 24-well plates and allowed to proliferate to near
confluence. They were then shifted to medium containing 1% FBS for
16-24 h without or with the compounds indicated. For the final 30 min
of incubation, culture medium was removed, and PBS containing the
respective test compounds was added. The PBS was then collected and
subjected to an ELISA specific for PGE2. Data are presented
as the mean ± S.E. of triplicate cultures from representative
experiments. A, effect of increasing concentrations of
exogenous arachidonate on PGE2 production in KAT-50 cells.
The concentrations of arachidonate indicated were added to cultures for
16 h. B, indomethacin (10 µM) or NS-398
(10 µM) was added to near-confluent KAT-50 cell cultures
in 24-well plates that were shifted to medium with 1% FBS and 5 µM arachidonate. The cultures were allowed to incubate
for 16 h.
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DISCUSSION |
We have found that human thyroid tissue can express high levels of
PGHS-2 under nonpathological states, as well as in the context of
several common neoplastic and inflammatory diseases. These observations
were unexpected and support questions concerning the currently held
view that the sole role of PGHS-2 involves mediation of
inflammation-related prostanoid production. Uncertainty over this
concept emerged several years ago from studies involving mice with
interruptions in the expression of PGHS-1 and -2. The resulting
phenotypes suggested a broader role for PGHS-2 (42-44). PGHS-2
expression observed on immunohistochemical staining in normal thyroid
tissue was primarily epithelial in location but also was found in
vascular cells. This finding in thyroid was unexpected because
precedent for such unprovoked PGHS-2 expression is limited to a very
few anatomic sites and cell types. Indeed, the kidney and central
nervous system have been shown to express PGHS-2 consistently under
basal, nonpathological conditions (19-21). Confirming our findings in
thyroid epithelium in situ is the strong corroborating
evidence generated in untreated KAT-50 cells in culture in which PGHS-2
mRNA, protein, and PGHS-2-dependent PGE2 production were also observed.
The molecular basis for the high level PGHS-2 expression in KAT-50
cells under basal culture conditions is not yet known. It does appear
to derive from unprovoked PGHS-2 promoter activity (Fig. 7) and from
relatively delayed mRNA degradation compared with that observed
previously in an endothelial cell line (12). The differences in
mRNA turnover could result from cell-specific expression of factors
interacting with the 3' untranslated region or from differences in the
PGHS-2 transcript itself. The finding of constitutive PGHS-2 in KAT-50
cells implies that the expression of this cyclooxygenase in thyroid
epithelium is driven by intrinsic phenotypic attributes of the
epithelium rather than by a factor(s) emanating from neighboring cells,
either those ordinarily resident in the gland or recruited in the
setting of disease. It suggests further that the PGHS-2 expressed
in situ in the thyroid is not a consequence of extrinsic
humoral or neurological influences. Indeed, KAT-50 cells under basal
conditions express substantial levels of IL-1
(Fig. 6) and blocking
the cellular actions of this endogenously generated cytokine with
either a neutralizing antibody or exogenous IL-1ra can markedly
attenuate PGHS-2 expression. Thus in these cells, IL-1
is
functioning as an autocrine factor. In contrast, KAT-50 cells do not
appear to express high levels of IL-1
or to utilize that cytokine in
the regulation of PGHS-2. Our finding that cycloheximide can block
constitutive PGHS-2 mRNA expression indicates the importance of
ongoing protein synthesis in driving the synthesis of this cyclooxygenase.
We have found that PGHS-1 mRNA, but not PGHS-1 protein, is
expressed by cultured KAT-50 cells. This is somewhat surprising in
light of the detectable PGHS-1 protein found by immunohistochemical means in situ in normal and disease thyroid tissue (Fig. 1).
Thus, it would appear that an important alteration in thyroid
epithelial cell phenotype associated with cultivation in culture may be
the loss of PGHS-1 translation. Interestingly, the PGHS mRNAs in
KAT-50 cells do not exhibit obvious differences from the transcripts expressed by fibroblasts with regard to electrophoretic mobility. Our
findings suggest that the PGHS-2 expressed in KAT-50 cells may have
assumed some of the metabolic functions ordinarily served by the PGHS-1
isoform in thyrocytes in situ. Obviously, it would be of
great interest to characterize the potentially different activities,
substrate utilization, and physiological functions of the two isoforms
in intact thyroid tissue. Particularly enlightening might be a
comparison between the subcellular localization of the PGHS isoforms in
thyroid tissue with that in tissues in which PGHS-2 expression is
confined to induced states.
The potent, synthetic glucocorticoid, dexamethasone, inhibited PGHS-2
expression when added to the culture medium of KAT-50 cells (Fig. 3).
Glucocorticoids have been shown to block the induction of PGHS-2 by
cytokines and growth factors and, in so doing, the production of
PGE2, in several cell types (2). Moreover, endogenous glucocorticoids exert negative tonic effects on the expression of this
enzyme in the intact animal, and when adrenal function is disrupted in
the mouse, exaggerated induction of PGHS-2 has been observed (18). The
finding that constitutive PGHS-2 is also attenuated by glucocorticoids,
as is the case in KAT-50 cells, suggests that under nonpathological
conditions, adrenal function may govern levels of cyclooxygenase
expression in the thyroid. To date, no glucocorticoid response elements
have been identified in the human PGHS-2 promoter (35), and it is
currently believed that the action of these steroids on the
NF-
B/I
B complex represents, at least in part, the molecular basis
for their down-regulation of cyclooxygenase expression and
PGE2 production. At least two NF-
B sites have thus far
been found upstream of the transcriptional start site in the PGHS-2
gene. These are located at
214/
204 and
447/
437 of the human
promoter (35, 36). Thus both elements are present in the two reporter
constructs utilized in our studies, both demonstrating substantial
basal activity, utilized in the transfection studies reported here.
Although they do not define the precise element(s) driving constitutive
PGHS-2 gene transcription in the KAT-50 cells, our findings do document
substantial basal activity of this promoter in these cells. Future
studies will be directed at generating a series of deletions by
site-directed mutagenesis along the human PGHS-2 promoter and defining
those elements that are relevant to the support of unprovoked PGHS-2 expression in KAT-50 cells.
PGHS-2 either is unexpressed or is found at extremely low levels in
most nondiseased tissues. With regard to isolated cells, the majority
fail to express detectable PGHS-2 mRNA or protein unless they are
treated with proinflammatory cytokines, serum, or growth factors. The
largest component of basal PGE2 production found in these
cells can be attributed to the activity of PGHS-1. With regard to human
fibroblasts from the synovium (13) or orbit (14), PGHS-2 is not
detectable under basal culture conditions. When the cells are treated
with IL-1
or leukoregulin, two inducers of PGHS-2, they express high
levels of the cyclooxygenase. Moreover, the increase in
PGE2 production observed in these fibroblasts following
cytokine treatment can be blocked with PGHS-2-selective inhibitors,
such as SC 58125 (46). The PGHS-2 up-regulation in fibroblasts and
endothelial cells appears to be a function of changes in both gene
transcription and mRNA stability (12, 14), whereas in other models,
an enhancement of gene transcription appears to predominate. Thus, the
regulation of PGHS-2 gene expression is complex and may be
cell-specific. Moreover, the anatomic region and state of health of the
tissue from which fibroblasts derive appear to determine the magnitude
of PGHS-2 induction (14, 47).
Overexpression of PGHS-2 has been linked to neoplasia of the colonic
epithelium. A number of reports have appeared recently, suggesting that
colon carcinomas exhibit high levels of PGHS-2 (48, 49), and some of
the cell lines derived from these lesions have been found to retain
this overexpression in vitro (50). Moreover, the high levels
of PGHS-2 are linked to changes in cell adhesion, metastatic potential,
and apoptosis (51, 52). The molecular basis for constitutive PGHS-2
expression in neoplastic cells is, in some cases, an increased rate of
gene transcription (53). Breast carcinoma and several breast cancer
cell lines and fibroblasts from these lesions exhibit particularly
robust PGHS-2 expression (54, 55). PGHS-1 overexpression may also be
related to neoplastic transformation. One report has demonstrated that
PGHS-1 expression in immortalized ECV endothelial cells can induce
neoplastic change (56). The chronic ingestion of nonsteroidal anti-inflammatory drugs is associated with a diminished incidence of
colon cancer (57). In animals, PGHS-2 inhibition with highly selective
agents has been found to decrease the numbers of colonic polyps in mice
with genetic mutations and enhanced susceptibility to intestinal
neoplasia (58, 59). Thus, intestinal expression of PGHS-2 may have
important pathogenic roles in the development of neoplastic disease of
the bowel. Our finding that normal thyroid epithelial cells in
situ express high levels of PGHS-2 raises questions concerning a
possible link between prostanoid biosynthesis and thyroidal neoplasia.
We have found high levels of PGHS-2 in malignant thyroid epithelium,
but our studies have not employed quantitative measurements of the
relative levels of cyclooxygenase in normal and diseased thyroid.
Clearly, such studies should now be undertaken to the extent that
current technology will allow.
One unanswered question that emerges from our observations relates to
the role of PGHS-2 in normal thyroidal function implied by constitutive
expression. Thyroid tissue is extremely vascular, and the factors
governing intrathyroidal blood flow/perfusion have not been fully
investigated. Prostanoids have been recognized for their regulatory
effects on blood flow in a variety of tissues (60) and thus locally
generated prostanoids in thyroid may prove important determinants of
thyroidal blood flow. We have demonstrated previously that
PGE2 is an important determinant of orbital fibroblast shape (61, 62), and the induction of PGHS-2 in those cells results
in a dramatic alteration of cellular morphology (14). The
PGE2 effects are mediated through an EP2 type
prostanoid receptor and result in a dramatic increase in cAMP levels
(62). Rapoport and Jones (63) reported that thyroid-stimulating hormone
induces a dramatic alteration in dog thyroid epithelial cell morphology in vitro. Although their report failed to link the
thyroid-stimulating hormone-dependent effects on morphology
to the activation of a particular signal transduction pathway, it is
possible that prostanoid generation, already associated with
thyroid-stimulating immunoglobulin action, might have been involved in
the shape change they observed. In preliminary studies, we have found
that thyroid-stimulating hormone can modulate PGHS-2 mRNA levels in
KAT-50 cells,3 raising the
possibility that the activated thyroid-stimulating hormone receptor
might serve to modulate intrathyroid cyclooxygenase expression. Another
possible role for PGE2 generated under both basal and
cytokine/growth factor-activated conditions might concern the
modulation of thyrocyte proliferation. In a very recent study, SC 58125 and dexamethasone could attenuate the growth inhibitory effects of
endothelin-1 and tumor necrosis factor
in human myofibroblastic hepatic stellate cells (25). The influences of these cytokines were
shown to be mediated through the induction of PGHS-2, which is
constitutively expressed in these cells (25). With regard to
thyrocytes, immunoglobulins from patients with Graves' disease have
been shown to stimulate arachidonate release through an activation of
the phospholipase A2 system and inositol
1,4,5-trisphosphate production in FRTL-5 cells and human thyrocytes
(28, 64). These IgGs elicit an enhanced rate of FRTL-5 proliferation,
an action that can be blocked by indomethacin (29). Although the cyclooxygenase products were not analyzed, nor was any attempt made to
identify the PGHS isoform involved, these findings do suggest that
cyclooxygenases might be crucial participants in thyrocyte signaling
related to cell proliferation. Moreover, Tahara et al. (65)
have demonstrated that in FRTL-5 rat thyroid cells, thyrotropin can
enhance cyclooxygenase-like activity and the formation of
prostaglandins. Our finding that a substantial fraction of the
PGE2 production in untreated KAT-50 cells could be
inhibited by NS-398, coupled with an absence of detectable PGHS-1
protein, implies that PGHS-2 represents the major functional
cyclooxygenase in these cells. We are currently conducting studies to
assess the effect of PGHS-2 interruption on the KAT-50 cell
phenotype.4
Another issue raised by our current findings concerns the potential
impact of cyclooxygenase inhibition on thyroid hormone biosynthesis.
The role of constitutive PGHS-2 expression in thyroid, as well as in
the other tissues where it has been found thus far, is uncertain.
However, the consequences of acute and chronic ingestion of
nonsteroidal anti-inflammatory drugs on thyroid function have never
been addressed. The effects of the currently available nonsteroidal anti-inflammatory drugs on basal PGHS activity may be subtle. The
recent availability of new PGHS-2-selective drugs, with their greatly
enhanced side effect profile, could allow more complete and sustained
inhibition of thyroidal PGHS-2 activity than achieved with currently
available drugs. Thus, it will be of great epidemiologic importance to
carefully monitor patients ingesting these newer drugs for any signs of
thyroidal dysfunction. The putative involvement of arachidonate
metabolites in mediating growth-stimulating actions of
thyroid-stimulating immunoglobulin suggests the possibility that PGHS-2
and its products may regulate thyrocyte cell division and apoptosis in
these cells.
PGE2 has been shown to influence several aspects of immune
function. For instance, the prostanoid can bias the commitment of naive
T lymphocytes (TH0) away from the TH1 phenotype
and toward that of TH2 (66, 67). PGE2 alters B
cell behavior (68) and participates in the activation of mast cells
(69). Prostanoids can modulate the expression of cytokines in
lymphocyte subsets (67) and thus can directly determine the molecular
environment present at sites of inflammation and wound repair. Because
the lymphocyte phenotype profile appears to determine, at least in part, the character of intrathyroidal immune responses, the products of
cyclooxygenases could condition immune responses occurring in that tissue.
A very recent paper by Sorli et al. (70) demonstrated that
PGHS-2 is the predominant cyclooxygenase isoform expressed in the
Syrian hamster islet cell line, HIT-T15, as well as in human and Syrian
hamster islets. Moreover, PGHS-2 is expressed constitutively in HIT-T15
cells. Treatment of these cells with exogenous IL-1 increased the
levels of PGHS-2 transiently. Unlike the KAT-50 cells and in
situ staining in thyroid, where PGHS-1 mRNA and protein, respectively, were detectable, the islet cells failed to exhibit any
evidence of PGHS-1 expression on either the mRNA or protein levels.
Those earlier studies also demonstrated surprisingly high levels of
NF-IL-6 and more modest levels of NF-
B in untreated cells expressing
constitutive PGHS-2. Another recent report by Kwon et al.
(45) has further characterized the expression of PGHS-2 in islet cells
and implicates the proteasome complex and NF-
B in this expression.
The potential implications of these findings in islet cells to the
pathogenesis of diabetes mellitus are considerable, given the proposed
modulatory role of prostaglandins on insulin release (39). Coupled with
our own observations reported here, we raise the possibility that
constitutive PGHS-2 expression might be considerably more widespread
than currently believed and could involve tissues and cell types that
have thus far not been inspected for cyclooxygenase expression. In
particular, other endocrine organs might represent sites where PGHS-2
is expressed under normal circumstances. Thus, the profile of
cyclooxygenase expression should be assessed in a wide array of these
tissues before any conclusions concerning the biological function of
these enzymes can be drawn. Further studies, including perhaps those involving animals in which the expression of the PGHS-2 gene is conditionally disrupted, either alone or in concert with that of
PGHS-1, will be necessary to define the precise role of this cyclooxygenase in healthy and diseased thyroid tissue.
 |
ACKNOWLEDGEMENTS |
We are grateful to Chris McBain, Heather
Meekins, and Sonia J. Parikh for expert technical assistance. We are
indebted to Dr. G. Drusano for helpful advice concerning the mRNA
decay calculations.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants EY08976 and EY11708 and by a Merit Review award from the
Research Service of the Department of Veterans Affairs.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: Division of
Molecular and Cellular Medicine (A-175), Department of Medicine, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Tel.: 518-262-5266; Fax: 518-262-5304.
2
T. J. Smith, unpublished observations.
3
D. Sciaky and T. J. Smith, manuscript in preparation.
4
H.-S. Wang and T. J. Smith, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
PGHS, prostaglandin-endoperoxide H synthase;
DRB, 5,6-dichlorobenzimidazole;
ELISA, enzyme-linked immunosorbent assay;
FBS, fetal bovine serum;
IL, interleukin;
NF-
B, nuclear factor-
B;
PBS, phosphate-buffered
saline;
PGE2, prostaglandin E2;
kb, kilobase pair(s);
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PMA, phorbol 12-myristate 13-acetate;
PAGE, polyacrylamide gel
electrophoresis.
 |
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