(Received for publication, April 17, 1997)
From the Labor Focus Research Group, Departments of
Pediatrics, ¶ Obstetrics and Gynecology, and
§ Biochemistry, Case Western Reserve University School
of Medicine, MetroHealth Medical Center, Cleveland, Ohio 44109
This report examines the effect of cell volume expansion on cyclooxygenase-2 (COX-2) mRNA expression, COX-2 protein expression, and prostaglandin E2 release from human amnion-derived WISH cells. Earle's balanced salts solution (EBSS) with limited NaCl concentration was utilized as the induction medium. COX-2 mRNA was elevated 6-fold in cells incubated for 1 h in hypotonic EBSS. COX-2 mRNA expression was not increased when raffinose or sucrose were used to reconstitute low NaCl. Actinomycin D blocked COX-2 mRNA increase by hypotonic stress, while cycloheximide enhanced COX-2 mRNA expression. COX-2 mRNA and protein concentrations increased as a function of decreasing media osmolarity and incubation time in hypotonic EBSS. Hypotonic EBSS induced a 3-fold increase in prostaglandin E2 release. WISH cells transiently transfected with a luciferase expression vector driven by the human COX-2 promoter for the COX-2 gene show a 3-fold increase in luciferase activity when incubated in hypotonic EBSS. COX-2 mRNA levels in primary human amnion cells were also increased by hypotonic stress. This study suggests that amnion cell COX-2 gene expression is regulated by cell volume expansion and/or increased plasma membrane tension.
Prostaglandins have a central role in regulating human parturition. Prostaglandin E2 (PGE2),1 the primary prostaglandin produced by fetal membranes during labor, may be directly involved in the initiation and maintenance of uterine contractions (1, 2). Untimely increases in prostaglandin biosynthesis early in gestation may be responsible for inducing preterm labor in some individuals (3). Although many hormones, growth factors, and cytokines have been reported to increase or decrease the synthesis and/or release of PGE2 in fetal tissues, the physiological factor(s) that up-regulates prostaglandin biosynthesis during parturition has not been identified.
Metabolism of arachidonic acid to prostaglandin H2 is a key and rate-limiting step in prostaglandin biosynthesis. The reaction is catalyzed by cyclooxygenase. Two isoforms of cyclooxygenase have been identified, designated as COX-1 and COX-2. Both enzymes have been characterized in considerable detail (for review, see Refs. 4-8). The genes for COX-1 and COX-2 are encoded on different chromosomes and have been sequenced. The gene for COX-1 is constitutively expressed, present in most tissues at low to nondeductible levels, and is generally considered to have "housekeeping" functions. In contrast, the gene for COX-2 has been characterized as an immediate early response gene. A wide range of mitogens, hormones, cytokines, and endotoxins increase the rates of COX-2 gene transcription and prostaglandin biosynthesis (4-8). COX-2 mRNA and COX-2 protein have been reported to increase near the onset of labor. A major site of PGE2 synthesis during labor occurs in the amnion (9, 10). COX-2 protein concentrations (11) and mRNA levels (10) are higher in amnion from women in labor versus patients not in labor. The underlying mechanism(s) leading to enhanced COX-2 mRNA expression in amnion during labor remains to be identified.
Fetal membranes clearly undergo mechanical stretching as a result of several processes during gestation, including fetal growth, increased amniotic fluid volume, and labor. Increased membrane tension of fetal cells may also occur as a direct result of cell volume expansion; human amniotic fluid osmolarity decreases as a function of advancing gestational age (12), raising the possibility that increased amnion cell plasma membrane tension might occur, in part, by changes in cell volume. Furthermore, as initially observed by Danforth and Hull (13) and as summarized by Alger and Pupkin (14), little if any amnion cell mitotic activity is observed in the latter part of gestation, and therefore, to accommodate the growing fetus, existing amnion cells must increase their size by stretching and hypertrophy. It has been recognized for some time that mechanical stretching of human cultured amnion cells increases the release of PGE2 (15). Although the biochemical mechanism by which mechanical stretching increases prostaglandin release from these tissues is largely unknown, it was recently reported that mechanical stretching of cultured rat glomerular mesangial cells induces a number of immediate early response genes including the gene for COX-2 (16). Based on this knowledge, we hypothesize that an increase in the volume of amnion cells and the resulting increase in plasma membrane tension induce COX-2 gene transcription and promote PGE2 release from fetal tissue. This study provides evidence that an increase in cell volume up-regulates COX-2 mRNA expression and elevates prostaglandin biosynthesis in amnion cells.
Amnion-derived WISH
cells were obtained from the American Type Culture Collection
(Rockville, MD) at passage 167. Tissue culture medium, designated as
DF10F, consists of a combination of Dulbecco's modified Eagle's
medium plus Ham's F-10 (50/50) supplemented with sodium bicarbonate
(14 mM), 15 mM Hepes buffer (pH 7.4) plus 10% fetal calf serum. DF10F contained the following antibiotics: penicillin (2 × 105 IU/liter), streptomycin (2 × 104 M), and ampicillin (25 mg/liter). For
each experiment, confluent WISH cell stock cultures were subcultured
from a Falcon T75 tissue culture flask into 60 × 15-mm Falcon
culture dishes (1.5 × 106 cells/dish) containing 5.0 ml of DF10F. Unless otherwise stated, cultures were incubated at
37 °C in humidified air containing 5% CO2, fed on day 3 with DF10F, and used for experiments on day 5. Under these growth
conditions, WISH cells reach confluence between the third and fourth
day of culture.
Cells cultured for 5 days in DF10F were washed with 2.0 ml of Earle's
balanced salts solution (EBSS). Induction medium (5.0 ml, equilibrated
to 37 °C), as defined under "Results," was added to cultures
that were then incubated at 37 °C in humidified air containing 5%
CO2. As described below, cells were then harvested for
preparation of mRNA or COX-2 protein. In some experiments, spent
incubation media were removed, frozen at 70 °C, and, as described
below, analyzed for prostaglandin concentrations.
Human amnion cells were cultured using a modification of the method of Okita et al. (17). Placentae and associated membranes were obtained from women undergoing repeat cesarean section. All tissue manipulations were performed using aseptic technique and sterile (0.2 µM filtered) solutions. Amnion was stripped from choriodecidua and washed successively in 200-ml changes of ice-cold Ca2+- and Mg2+-free phosphate-buffered saline containing gentamicin (50 µg/ml) until clear of blood. Tissue was minced in 50 ml of TEP buffer (0.05% trypsin (SIGMA, Type II), 0.02% EDTA in phosphate-buffered saline) and then incubated at 37 °C for 20 min in a shaking water bath. The preparation was filtered through 2-mm stainless steel wire gauze. The filtrate was discarded, and the tissue fragments were recombined with 150 ml of TEP and incubated for 90 min. Following incubation, the cell suspension was combined with 450 ml of ice-cold phosphate-buffered saline, shaken for 15 s, and filtered sequentially through 2- and 0.7-mm stainless steel wire gauze. The filtrate was centrifuged at 1000 × g for 8 min at 4 °C. Pelleted cells were resuspended in Earle's minimal essential medium, plated in 150-mm tissue culture dishes, and then incubated at 37 °C in 5% CO2 at 95% relative humidity. Medium was changed initially at 24 h and subsequently at 48-h intervals. Cells were grown to confluence and used directly.
Preparation of RNAInduction media were removed from
dishes, and 2.5 ml of RNA lysis buffer (4.0 M guanidine
isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5%
sarcosyl, and 0.1 M 2-mercaptoethanol) was added to cell
cultures. Cultures were allowed to stand in the lysis buffer for 5 min
at room temperature and then mixed repeatedly by pipette. Cell lysates
were transferred to Falcon number 2059 test tubes and frozen at
70 °C until preparation of RNA. Frozen cell lysates were thawed
and passed three times through an 18-gauge needle. The sample (2.1 ml)
was layered on a 2.5-ml pad of 5.7 M CsCl containing 0.1 M EDTA and then centrifuged at 35,000 rpm for 18 h at
18 °C employing a Beckman SW 50.1 rotor.
An aliquot from each RNA sample was diluted into TE buffer (10 mM Tris plus 0.1 mM EDTA, pH 7.4), and the
concentration of RNA was estimated spectrophotometrically as described
previously (18). Specific amounts of RNA (10 µg/well for Northern
analysis and 5 µg/well for dot blots) from each sample were aliquoted
into microcentrifuge tubes. Sufficient diethylpyrocarbonate-treated water was added to each sample to provide the same RNA concentrations throughout all tubes. For Northern analyses, RNA precipitation solution
(0.1 volume of 3 M sodium acetate, pH 5.0, followed by 2.5 volumes of 100% ethanol) was added to the samples, which were then
stored at 70 °C until used. For dot blot assays, RNA samples were
processed as described below.
For Northern analysis, RNA samples were pelleted from the precipitation solution by centrifugation (12,000 × g for 15 min at 5 °C) and then dried by vacuum. Pellets were dissolved into 10 µl of denaturing solution (5 µl of formaldehyde, 2 µl of formamide, 1 µl of 10 × MOPS buffer (0.2 M MOPS), 0.1 M sodium acetate, 10 mM EDTA, pH 7.0), and 2 µl of H2O) plus 1 µl of ethidium bromide (400 µg/ml H2O). The samples were heated for 10 min at 65 °C and then chilled on ice. Samples were electrophoresed in formaldehyde-agarose gels (1.0% agarose (w/v), 6.6% formaldehyde (v/v) in 1 × MOPS buffer). Electrophoresis was carried out in 1 × MOPS buffer overnight (0.15 V/cm2). RNA was transferred by capillary action to Nytran filters overnight employing 10 × SSC (1.5 M sodium chloride, 150 mM sodium citrate, pH 7.0) as the transfer buffer. The Nytran filters were air-dried and then baked at 80 °C for 1.5 h. As previously demonstrated (19), utilization of ethidium bromide under the above denaturing conditions provided direct evidence that (a) approximately equal amounts of RNA from different cell cultures were applied to gels for electrophoresis, (b) RNA integrity was maintained during sample preparation and electrophoresis, and (c) the transfer of RNA from gel to filter was complete.
RNA dot blots were performed with a VacuDot-VS manifold (American Bionetics). Nytran filters were prewet with 6 × SSC prior to applying samples. RNA solutions were diluted with 4 volumes of denaturing solution and then heated at 65 °C for 15 min. Solutions of denatured RNA were chilled on ice, diluted with 1.5 volumes of 6 × SSC, and then loaded into manifold wells. The samples were allowed to drain by gravity for 30 min prior to applying a vacuum. Each well was vacuum-washed twice with 400 µl of 6 × SSC. Filters were air-dried and then heated at 80 °C for 1.5 h. Filters from both Northern blots and dot blots were then incubated for 2 h at 42 °C in prehybridization buffer as described previously (20).
The 32P-labeled probe for COX-2 mRNA was generated with a random primed labeling kit (Amersham International plc, Buckinghamshire, United Kingdom). The substrate was the 1.2-kilobase COX-2 cDNA insert purified from plasmid pcDNAhCOX-2. After prehybridization, the 32P-labeled COX-2 cDNA probe was denatured and then added directly to the prehybridization buffer (1.0 × 106 dpm/ml). Filters were incubated overnight at 42 °C. They were then incubated twice in 6 × SSC buffer plus 0.5% SDS and once in 1 × SSC plus 0.1% SDS for 15 min at room temperature. The final wash was carried out in 1 × SSC plus 0.1% SDS for 30 min at 56 °C.
Autoradiography and COX-2 mRNA AnalysisNytran filters
from Northern blots and dot blots were exposed to Kodak X-Omat RP film
at 70 °C employing Lighting Plus intensifying screens.
Autoradiographs of 32P-hybridized RNA dot blots were
scanned with a Microtek MSF-300GS image scanner (Microtek, Torrance,
CA) that was linked to a Macintosh IIsi computer. The image generated
by the Microtek grayscale scanner was captured by Image Studio software
(Letraset, Paramus, NJ) and then analyzed for intensity of grays
relative to background by Scan Analysis software (Biosoft, Milltown,
NJ). Quantitation of image intensities on film was carried out at less
than maximal densities as described previously (20). When an image from
a given dot blot was determined to be overexposed, autoradiography was
repeated employing a shorter exposure time.
Monolayers of WISH cells were
suspended in 0.5 ml of lysis buffer (20 mM Hepes (pH 7.2),
120 mM NaCl, 1% Triton X-100, 5 µg/ml aprotinin, 10 µg/ml antipain, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, and 0.1 mM phenylmethylsulfonyl fluoride) and frozen at 70 °C. Cell homogenates were thawed on ice and maintained at 4 °C
throughout manipulation. Homogenates were sonicated for 10 s (Heat
Systems-Ultrasonics, Inc., model W225R with microtip), setting 4, 100%
duty cycle, and then cleared by centrifugation at 12,000 × g for 5 min at 4 °C. Aliquots (25 µl) of homogenates
were subjected to polyacrylamide gel electrophoresis on 8% gels. Gels
were equilibrated in 20 mM Tris, 190 mM
glycine, 20% methanol, pH 8.3 overnight at 10 °C. Resolved proteins
were semi-dry transferred for 1 h at 10 V (Bio-Rad, Melville, NY)
to Hybond N+ (Amersham Corp.). Western blots were blocked in wash
buffer plus bovine serum albumin (BSA) (10 mM Tris, pH 7.2, 100 mM NaCl, 0.2% Tween-20, containing 1% fatty-acid free
BSA) overnight at 4 °C prior to incubation with COX-2 antibody (1:500 dilution in wash buffer plus BSA) for 1 h at 25 °C.
Blots were washed in three changes of wash buffer at 10-min intervals and then incubated with a 1:2000 dilution of anti-mouse IgG-horseradish peroxidase conjugate in wash buffer plus BSA overnight at 4 °C. Following incubation, blots were washed as before prior to signal generation by ECL (Amersham) and visualized by a 10-s exposure to Kodak
X-Omat AR film.
WISH cells were plated in 3.8-cm2
wells at a density of 5.0 × 105 cells, 2.0 ml of
DF10F. After 24 h in culture the medium was removed, and cells
were liposome-mediated transfected employing the following: 50 µg of
LipofectAMINE (Life Technologies, Inc.), 10 µg of the COX-2 pXP1
luciferase reporter vector, 2 µg of the secreted alkaline phosphatase
genetic reporter system (pSEAP-Control vector;
CLONTECH, Palo Alto, CA), and 1.0 ml of DF10F
lacking serum and antibiotics. Cells were incubated for 16 h in
the initial transfection medium, after which an additional 1.0 ml of
DF10F containing 10% fetal calf serum was added to each plate. The
transfection medium was removed after 24 h, and cells were then
incubated for an additional 48 h in DF10F plus 10% fetal calf
serum minus antibiotics. Induction media, as defined under
"Results," were added to plates, and cells were incubated for
designated times at 37 °C in 5% CO2. For alkaline
phosphatase assays, induction media (500 µl) were removed from each
plate and then centrifuged at 12,000 × g for 5 min at
10 °C. The supernatants were frozen at 70 °C until analysis by
chemiluminescent detection using a commercial system
(CLONTECH) and a 96-well format. Samples were
exposed to x-ray film for various time periods to assure that detection
was within the linear range of the film. For luciferase activity, cells
were scraped into 200 µl of luciferase assay lysis buffer (Promega)
and then sonicated (Heat Systems-ultrasonics Inc.) for 5 s on ice
at 50% output. Samples were centrifuged at 12,000 × g for 5 min at 10 °C. Luciferase activity was determined
using a commercial kit (Promega) by scintillation counting per the
manufacturer's instructions.
Prostaglandin antibodies and anti-mouse IgG-horseradish peroxidase conjugate were from Sigma. COX-2 antibodies were from Transduction Laboratories (Lexington, KY). PGE2 was determined by radioimmunoassay assay using a commercial antibody and following the supplier's protocol (Sigma). Media osmolalities were determined by vapor pressure employing a Wescor 5500 Vapor Pressure Osmometer (Wescor Inc., Logan, Utah). The plasmid pcDNAhCOX-2, which contains mouse COX-2 cDNA (21), was a generous gift from Dr. Timothy Hla (American Red Cross, Rockville, MD). The vector containing the human COX-2 promoter-luciferase reporter construct (22) was a generous gift from Dr. Lee-Ho Wang (University of Texas, Houston).
COX-2 mRNA
expression was initially examined in WISH cells incubated for 1 h
in hypotonic versus isotonic EBSS. Relative to the
concentration of COX-2 mRNA from cells incubated in isotonic EBSS,
COX-2 mRNA expression was markedly elevated in hypotonically stressed cells (Fig. 1, lanes
2, 5, and 8 versus lanes 3, 6,
and 9, respectively). The 32P-labeled COX-2
cDNA probe hybridized to three mRNA species of approximately
5.8, 4.8, and 3.4 kb. All three mRNA species were elevated in
hypotonically stressed cells. In contrast to COX-2 mRNA expression,
no change in the level of COX-1 mRNA was detectable by Northern
analysis employing a cDNA-specific probe for COX-1 (not shown).
The relative concentration of basal level COX-2 mRNA from untreated
cells (i.e. cells harvested directly from spent tissue culture growth medium, DF10F) was also determined (Fig. 1, lanes 1, 4, and 7). Basal COX-2 mRNA levels
were consistently less than levels from WISH cells incubated in
isotonic EBSS (compare lanes 1, 4, and
7 to lanes 2, 5, and 8,
respectively). During the course of these studies it was determined
that, due to evaporation, the osmolarity of fresh DF10F tissue culture
medium increased from 290-300 mosmol/liter to 315-320 mosmol/liter
after 48 h of incubation. The osmolarity of standard isotonic EBSS
routinely assayed between 280 and 290 mosmol/liter and, therefore,
cells cultured for 48 h in DF10F medium and then incubated in
isotonic EBSS were subjected to a decrease in osmolarity of between 25 and 40 mosmol/liter. Thus, shifting cells from 48-h spent tissue
culture media that has become moderately hypertonic due to evaporation
to standard isotonic EBSS is sufficient to increase COX-2 mRNA
expression. As shown in Fig. 3, very little if any difference in COX-2
mRNA concentrations from untreated cells versus isotonic
EBSS-treated cells were observed when the osmolarity of EBSS was
increased by 30 mosmol/liter with 30 mM raffinose.
As determined by autoradiography and grayscale scanning of RNA dot blots hybridized to the 32P-labeled COX-2 cDNA probe, the relative concentration of COX-2 mRNA from WISH cells incubated for 1 h in hypotonic EBSS was elevated approximately 6-fold above the COX-2 mRNA concentration from cells incubated in isotonic EBSS (Table I). Numerous reports have shown that EGF is a potent inducer of COX-2 gene transcription in a variety of biological systems, including human amnion cells. As a positive control in this study, and to compare the relative potency of hypotonic stress to a recognized inducer of COX-2 gene expression, the effect of EGF was also determined on WISH COX-2 mRNA expression. The relative concentration of COX-2 mRNA in cells incubated in isotonic EBSS plus EGF was elevated approximately 10-fold higher than that in cells incubated in isotonic EBSS (Table I).
|
Hypotonic media in Fig. 1 and Table I were
formulated by decreasing the amount of NaCl in EBSS. A previous study,
employing rat papillary collecting tubule cells, reported that
PGE2 was induced in this system by a reduction in
extracellular Na+ (23). To determine if elevated WISH cell
COX-2 mRNA expression is related to cell volume expansion or,
alternatively, related to a reduction in extracellular Na+
and/or Cl, cells were incubated in isotonic EBSS in which
116 mosM sucrose or raffinose was substituted for 116 mosM NaCl (Fig.
2A). Only cells incubated in
hypotonic medium were characterized by increased COX-2 mRNA
expression. In the same experiment, the concentration of released
PGE2 was determined in induction media from WISH cells (Fig. 2B). PGE2 was also elevated only in cells
incubated in hypotonic EBSS.
Effects of Actinomycin D and Cycloheximide on COX-2 mRNA Expression
The effects of actinomycin D and cycloheximide on elevated COX-2 mRNA resulting from hypotonic stress were determined by dot blot analysis (Fig. 3). The addition of actinomycin D to hypotonic EBSS completely suppressed COX-2 mRNA concentrations to levels observed for untreated cells and cells incubated in isotonic EBSS. In contrast, the relative concentration of COX-2 mRNA in hypotonically stressed cells was not reduced by the addition of cycloheximide to hypotonic EBSS.
Kinetics of COX-2 mRNA ExpressionCOX-2 mRNA
expression was examined as a function of decreasing medium osmolarity
(Fig. 4A). The relative
concentration of COX-2 mRNA was characterized by a gradual increase
as the osmolarity was reduced from 279 mosM to 200 mosM. A more pronounced increase in COX-2 mRNA
expression was evident when the osmolarity was shifted from 200 to 178 mosM, the lowest osmolarity tested.
The relative concentration of COX-2 mRNA was also examined as a function of incubation time in hypotonic medium (Fig. 4B, open circles). A change in WISH cell COX-2 mRNA levels resulting from hypotonic induction medium (161 mosM EBSS) was detected within 30 min, the earliest time period examined. COX-2 mRNA levels remained elevated for an additional hour and then declined to near basal levels between 2.5 and 3.0 h.
Hypotonic Stress and COX-2 Protein ExpressionRelative to
cells incubated in isotonic EBSS, cells subjected to hypotonic EBSS
(176 mosM and 218 mosM) were characterized by
increased COX-2 protein concentration (Fig.
5). Two COX-2 protein bands of 72 and 74 kDa were detected, an observation that is consistent with the sizes of
COX-2 protein reported for other systems (24). At 176 mosM,
COX-2 protein appears to be maximally expressed between 1 and 2 h,
and then it falls to basal levels between 3 and 6 h, a pattern
that closely parallels the increase in COX-2 mRNA expression when
cells are incubated in 161 mosM EBSS (Fig. 4B).
When incubated in a less pronounced hypotonic EBSS medium (218 mosM), the increase in COX-2 protein expression was delayed
for at least 2 h, increasing between 3 and 6 h, and remained
elevated for several hours prior to returning to basal levels between 6 and 18 h (Fig. 5).
Response of COX-2 Promoter-Luciferase Construct to Hypotonic Stress
To ascertain the effect of hypotonic stress on COX-2 gene
expression, WISH cells were transiently transfected with a human COX-2
promoter-luciferase construct. This reporter construct contains an
899-base pair fragment of the human promoter for the COX-2 gene,
progressing 5 from the ATG start site, and coupled to the Pxp1
luciferase expression vector (22). Relative to cells incubated in
isotonic EBSS, cells incubated in hypotonic EBSS were characterized by
approximately a 3-fold increase in luciferase activity (Fig. 6). The level of luciferase induction
observed for cells incubated in isotonic EBSS plus EGF was
approximately 6-fold above cells incubated in isotonic EBSS (Fig.
6).
Hypotonic Stress and COX-2 Expression in Primary Human Amnion Cells
Because WISH cells are immortal and therefore transformed,
it cannot be assumed that the activation of a given signal transduction pathway in this cell line is applicable to normal human amnion cells.
Previous studies of primary human amnion cells in culture have show
that they retain many of the biochemical and morphological properties
of amnion cells studied immediately after parturition (17). For this
reason the effect of cell volume expansion on COX-2 mRNA expression
was determined on primary human amnion cells prepared from placentae of
women undergoing repeat cesarean section (Fig.
7). Relative to cells incubated for
1 h in isotonic EBSS, COX-2 mRNA was markedly elevated in
cells incubated in hypotonic EBSS. The increased concentration of COX-2
mRNA in primary human amnion cells resulting from hypotonic stress
was approximately 50% of that induced by EGF (Fig. 7), a pattern that
is consistent with WISH cell COX-2 mRNA expression induced by EGF
versus hypotonic stress (Table I).
The central finding in this study is that hypotonic stress increases prostaglandin biosynthesis in amnion cells. Results in this report demonstrate that COX-2 mRNA expression, the amount of COX-2 protein, and the release of PGE2 from WISH cells originally derived from amnion are all enhanced by cell volume expansion.
The expression of three apparent COX-2 mRNA species in hypotonically stressed WISH cells was an unexpected finding (Fig. 1). Most earlier studies of COX-2 mRNA expression, employing a wide variety of biological systems, reported the presence of a single COX-2 mRNA species ranging in size from 4.2 to 4.8 kb (4-8). Recently, in an extensive study of COX-2 mRNA isoforms induced by cytokines in human lung and kidney cells (25), three isoforms of COX-2 mRNA were detected: two major isoforms of 4.6 and 2.8 kb and a minor species of 4.1 kb. In the same study it was shown that the different isoforms are a product of alternative polyadenylation and, surprisingly, that the 2.8-kb COX-2 mRNA had a significantly longer half-life than the 4.6-kb species. In the current study, as determined by Northern analysis, two major COX-2 bands of 5.8 and 3.4 kb were detected, with a less pronounced band of 4.8 kb (Fig. 1). In a previous communication, interleukin-18 induced a single COX-2 mRNA species of 5.5 kb in primary human amnion cells from term placenta (26). The physiological significance of several COX-2 mRNA bands in WISH cells is unclear. To our knowledge, the large 5.5-5.8-kb species of COX-2 mRNA has only been detected in WISH cells and primary amnion cells. It remains to be established if the various COX-2 mRNA species in hypotonically stressed WISH cells are a result of alternative polyadenylation or alternative splicing. Knowledge of potential changes in the turnover rate of COX-2 mRNA, as a result of increased cell volume and/or increased membrane tension, may provide important information concerning the regulation of prostaglandin metabolism in amnion cells.
The observation that hypotonic stress induces luciferase in WISH cells transfected with a human COX-2 promoter-luciferase vector (Fig. 6), coupled with the knowledge that actinomycin D prevents hypotonic stress from increasing COX-2 mRNA levels (Fig. 3), suggests that changes in prostaglandin biosynthesis resulting from cell volume expansion are due to an increase in the rate of COX-2 gene transcription. Hypotonic stress induced a large increase in COX-2 mRNA levels within 30 min after treatment (Fig. 4B). The increase in COX-2 mRNA associated with cell volume expansion was not suppressed by cycloheximide (Fig. 3). Collectively, results in this study demonstrate that the gene for COX-2 acts as an immediate early response gene when stimulated by cell volume expansion. This suggestion is consistent with previous reports, employing a variety of biological systems, that classified the gene for COX-2 as an immediate early response gene following treatment with different growth factors, mitogens, and cytokines (4-8).
The signal transduction pathway(s) that is activated in higher eukaryotes by cell volume expansion and leads to increased gene expression has not been identified. Nevertheless, recent studies employing yeast mutants have demonstrated that hypotonic stress activates the PKC1 pathway (27), one of four recognized yeast mitogen-activated protein kinase pathways (MAP kinase pathway). These studies demonstrated that a functional PKC1 pathway is required for the survival of yeast in a hypotonic environment. In contrast to hypotonic stress, hypertonic stress activates a different MAP kinase pathway in yeast, designated as the HOG pathway. This pathway is required for survival of yeast in a hypertonic environment. Interestingly, hypertonic stress of Chinese hamster ovary cells produces a marked increase in Jnk 1 (28), an enzyme similar to the yeast protein kinase HOG1. Furthermore, Jnk 1 was able to rescue yeast mutants lacking functional HOG 1 from hypertonic shock. Since yeast and mammalian systems appear to have similar MAP kinase pathways and these pathways may be important in cell volume regulation (27-29), it is possible that a PKC1-like pathway exists in amnion cells and that this pathway is activated by cell volume expansion. The end product of this pathway may, in turn, modify a factor that increases the rate of COX-2 gene transcription.
Cell volume expansion and membrane stretch have been shown to modify
the expression of a wide variety of genes in a number of diverse
biology systems. Previous studies have reported that increased cell
volume resulting from hypotonic stress modifies the concentration or
activity of a number of factors that impact, directly or indirectly, on
the transcription rate of specific genes (for a review, see Ref. 30)
including, for example, intracellular calcium, cyclic AMP, tyrosine
kinases, inositol 1,4,5-trisphosphate, and protein kinase C. Although
the mechanism by which cell volume expansion increases amnion cell
COX-2 gene transcription remains to be characterized, the promotor
region for the human COX-2 gene has been shown to contain a wide
variety of potential regulatory elements, including CRE, NF-B, Sp1,
and AP2 sites (22). Recently, what appears to be a novel putative
cis-element in the promoter region of rabbit aldose reductase has been
shown to be necessary for increased expression of this gene during
hypertonic stress (31). Perhaps a novel regulatory element, yet to be
identified, is essential for regulating gene expression as a function
of cell volume expansion and/or increased membrane tension.
It has been recognized for more than 35 years that prostaglandins provoke uterine contractions (for a historical review of prostaglandins and uterine contraction, see Ref. 32). Within the past 10 years, a vast array of studies have documented that prostaglandins have an important role in human parturition. It is well established that amnion is a major site of prostaglandin biosynthesis. Approximately 10 years ago it was documented that mechanical stretching of cultured human primary amnion cells increases the release of PGE2 (15). Recent studies have shown that prostaglandin biosynthesis increases in amnion prior to labor, and the increase is associated with elevated cyclooxygenase expression (10, 11, 33), a rate-limiting step in prostaglandin biosynthesis (4-8). Although more than 30 hormones, mitogens, and cytokines have been shown to increase prostaglandin production, the mechanism by which increased PGE2 biosynthesis occurs in fetal tissues remains unclear. Studies in this report raise the possibility that an increase in cell membrane tension induced by cell volume expansion and perhaps cell membrane stretching up-regulates the rate of COX-2 gene transcription, increasing PGE2 biosynthesis and release in cells derived from human amnion. These observations raise the additional possibility that increasing mechanical forces resulting from increased cell volume and/or membrane stretch during gestation may be a critical factor in the initiation of labor.
We are grateful to Dr. Mary Lou Kumar for making available certain tissue culture equipment and Dr. John Harris for suggestions in preparation of this manuscript.