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Original Studies |
Is Up-Regulated by Pregnancy Serum1
Departments of Obstetrics, Gynecology, and Reproductive Sciences (L.L.W., R.N.T.), Pharmaceutical Chemistry and Cellular and Molecular Pharmacology (T.S.S.), Stomatology (Y.Z.), and Graduate Program in Biophysics (E.C.P.), University of California, San Francisco, California 94143; and Department of Obstetrics and Gynecology, Harvard Beth Israel-Deaconess Hospital (K.-H.L.), Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Dr. Leslie L. Waite, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, California 94143.
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Abstract |
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(PPAR) in tissues and cells
relevant to human pregnancy. We found that PPAR is expressed in
placental cytotrophoblasts in vivo and in trophoblasts
(primary and choriocarcinoma cells) and fetal endothelial cells
in vitro. We characterized primary cytotrophoblasts and
two cell lines with which to study PPAR regulation in human
pregnancy. Like primary cytotrophoblasts, the choriocarcinoma cell line
JEG-3 has endogenous PPAR expression. Normal positive and negative
PPAR regulation was observed in the latter cells. We also
created a new JEG-3-derived cell line (EP-JEG) by stable insertion
of a PPAR response element-luciferase reporter gene construct.
Together, these cell lines are useful for studying PPAR expression
and activation in human trophoblasts. We examined PPAR regulation in
these cells by human serum and found that PPAR protein expression
and activation are dramatically increased by sera from pregnant women.
Preliminary characterization of the regulatory principle(s) is
consistent with a prostanoid or fatty acid derivative. The results
suggest that increased activation of PPAR may play an important role
in maternal metabolism during human pregnancy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, NUC1 (also known as PPARß or
-
in other species), and PPAR. These receptors regulate
lipid and glucose homeostasis and metabolism. In particular,
PPAR has been shown to be a major regulator of adipogenesis (3, 4, 5, 6)
and is a target of antidiabetic drugs of the thiazolidinedione (TZD)
family. Activation of PPAR, presumably by PGs or other fatty
acid metabolites, leads to target gene transcription via a consensus
PPAR response element (PPRE). These PPREs are activated by a
heterodimer of PPAR with the retinoid X receptor (7). One
specific activator of PPAR is
15-deoxy-
12,14-prostaglandin
J2 (15dJ2), a derivative of
PGD2 (8, 9). Several studies also have shown that
the cytokine tumor necrosis factor- (TNF) can inhibit the effects
of PPAR in vitro and in vivo (10, 11, 12, 13). Thus,
these two compounds are useful tools to examine positive and
negative regulation, respectively, of the PPAR receptor.
Recent work suggests that PPARs play a role in hemochorial
placentation. For example, studies by Barak et al. (14)
demonstrate that PPAR is essential for normal placental development
in mice. Embryos without this gene show massive placental defects that
can be rescued by restoration of the PPAR gene via chimeras. Lim
et al. (15) found that prostacylin synthesis is essential
for implantation and decidualization in mice, and that its actions
appear to involve PPAR. Although substantial placental anatomical
differences exist between mice and humans, in vitro studies
using human cells complement the findings in mice. Keelan et
al. (16) and Matsuo and Strauss (17) have identified messenger RNA
(mRNA) for PPAR and NUC1, respectively, in JEG-3 cells, a
choriocarcinoma cell line derived from placental trophoblasts.
Dramatic maternal physiological changes in human pregnancy necessitate modified regulation of glucose and lipid metabolism. During normal pregnancy, maternal energy and lipid metabolism are profoundly altered (18). The developing fetus uses glucose as its predominant energy source, which puts a continuous demand on the mother to provide this substrate (19). Problems with sugar metabolism, such as hypoglycemia or, alternatively, gestational diabetes, are not uncommon during pregnancy. Lipid metabolism also changes in pregnancy. Levels of circulating free fatty acids are in the normal range during most of pregnancy, but rise dramatically during the final weeks of pregnancy and drop precipitously at term (20). Additionally, maternal triglyceride levels triple during pregnancy (21). PG metabolism also is altered, with levels of vasorelaxants such as prostacylin increasing, whereas vasoconstrictive PG levels decrease (22, 23, 24). Failure of these alterations to occur may lead to pregnancy complications (e.g. preeclampsia) (25, 26).
In the current study we report the expression and localization of
PPAR protein in placental cytotrophoblasts in vivo.
Moreover, we have characterized two human trophoblast cell lines for
the study of PPAR regulation in pregnancy. Using these cell lines,
we have determined that human pregnancy serum contains a factor(s) that
up-regulates the expression and activation of PPAR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum was prepared from freshly clotted whole blood collected under sterile technique and frozen at -70 C within 12 h of collection. Serum samples from men, nonpregnant reproductive age women (20–40 yr), and pregnant women (36–40 weeks gestation) were collected. All samples were drawn from nonfasting individuals. Six samples from each group were used in each experiment. In experiments in which pools were used, six individual samples were pooled for each group.
Cell culture
All cells were cultured at 37 C in 5% CO2. JEG-3 cells (obtained from American Type Culture Collection, Manassas VA), JAr cells (a gift from Dr. Susan Fisher), and EP-JEG cells were cultured in Eagle’s MEM with Earle’s salts containing 10% FBS, penicillin (100 U/mL), streptomycin (100 µg/mL), and gentamicin (50 µg/mL). Human umbilical vein endothelial (HUVE) cells were isolated according to previously described protocols (33) and cultured on 1% gelatin-coated plates in DMEM/Ham’s F-10 (50:50) plus the following: endothelial cell growth supplement (100 µg/mL), glutamine (292 µg/mL), pyruvate (110 µg/mL), FBS (20%), and antibiotics as described above. Primary cytotrophoblasts from first and second trimester human placentas were isolated and cultured as previously described (34, 35).
Generation of stable transfectants
Stable transfectants expressing integrated copies of the
reporter plasmid PPRE3-thymidine kinase
(TK)-luciferase (LUC) in JEG-3 cells were obtained using previously
described procedures (36). Briefly, a plasmid containing the reporter
gene construct PPRE3-TK-LUC and a second plasmid
containing a neomycin resistance marker were linearized with
XmnI and introduced into JEG-3 cells by electroporation.
Cells were maintained under continuous G418 selection for 30 days, and
48 colonies were isolated. Once isolated, colonies were grown in medium
without G418. Of these 48 lines, 10 showed a measurable luciferase
response to 10 µmol/L rosiglitazone. In further testing, 4 of these
showed both low relative basal signal magnitude and robust activation
by rosiglitazone relative to the vehicle alone. After checking the
responses of these 4 lines to other known PPAR ligands (including
15dJ2), 1 line, termed EP-JEG, showed superior
consistency of response among experiments and was chosen for the
studies described here.
Immunofluorescence studies
Immunofluorescence histochemistry on frozen human placental bed
sections (20 weeks gestational age) was performed as previously
described (37) using anti-PPAR (sc-1985, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-PPAR antibody
(sc-7273, Santa Cruz Biotechnology, Inc.) at a dilution of
1:100.
RT-PCR
Total RNA from JAr, JEG-3, primary placental cytotrophoblasts,
and HUVE cells was isolated from
106–107 cells using Trizol
(Life Technologies, Inc.). Testis RNA was a gift from Dr.
Ximena Ares. One microgram of each RNA was reverse transcribed in a
20-µL reaction containing 4 mmol/L MgCl2;
1 x PCR buffer (Life Technologies, Inc.); 1 U
reverse transcriptase, 100 µmol/L each of deoxy (d)-ATP, dCTP, dGTP,
and dTTP; and 2.5 µmol/L random hexamers. The entire 20-µL reaction
was then subjected to PCR amplification. PCR reaction conditions were
similar to those described above, with Taq and Taqstart
antibody [0.5 U Taq equivalent, according to CLONTECH Laboratories, Inc. (Palo Alto, CA), instructions] replacing
reverse transcriptase. The final volume of the PCR reaction was 100
µL. Sequences of the PPAR-specific primers were: upstream,
5'-AACTGC AGGGTT GACACA GAGATC GC-3'; and downstream, 5'-GGAATT CTGCAA
CCACTG GATCTG TTC-3'. These sequences are based on those used in the
mouse (38) and amplify the first 183 bp of the human PPAR1 sequence
while adding PstI and EcoRI restriction sites,
upstream and downstream, respectively, to the PCR products. Cycle
parameters for PCR amplification were 94 C for 5 min; 8 cycles of 94 C
for 20 s, 57 C for 20 s, and 72 C for 80 s; and 25
cycles of 94 C for 20 s, 62 C for 20 s, and 72 C for 80
s. A final extension round (72 C, 7 min) was used to maximize
complete product formation. Ten microliters of each reaction were
removed and incubated with HincII enzyme to allow digestion.
These digested samples and 10 µL of the corresponding undigested PCR
product were mixed with sample buffer and loaded onto a 3%
Nu-Sieve gel (FMC, Rockland, ME). After electrophoresis, gels were
stained with ethidium bromide and photographed.
Treatment of cells with activators and inhibitors of PPAR
JEG-3 or EP-JEG cells were treated with trypsin to allow removal from flasks. Cells were pelleted, and trypsin medium was removed. Cells were then resuspended and counted using a hemocytometer and replated onto 12-well tissue culture plates at a density of 1.6 x 105 cells/well. After overnight incubation, media were removed, and cells were rinsed with PBS. After removal of PBS and transient transfection of JEG-3 cells, cells were treated in triplicate for 24 h with the media containing the compounds described in the text before analysis as described below.
Transient transfection
JEG-3 cells were transfected using Superfect (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. The transfection time was 2.5 h with 280 ng DNA/well. A mixture of 5 ng PPRE-luciferase plasmid (7)/1 ng TK-Renilla luciferase plasmid (Promega Corp., no. E2241, internal control) was used.
Luciferase activity assay
Cells were lysed with 250 µL of the appropriate lysis buffer (see below), and 20 µL (JEG-3) or 40 µL (EP-JEG) lysate were used for luciferase analysis. JEG-3 cells were analyzed using a dual luciferase protocol (Promega Corp.) that allows independent measurement of both PPRE3-TK-firefly luciferase and TK-Renilla luciferase. Firefly luciferase activity was expressed as a ratio of Renilla activity for all calculations. For EP-JEGs, firefly luciferase activity was measured using a single luciferase protocol according to the manufacturer’s instructions (Roche, Indianapolis, IN). Lysis buffer alone also was analyzed, and this value was subtracted from sample values before calculating relative activation. In both JEG-3 and EP-JEG cells, luciferase activity in treated cells was normalized to activity in cells incubated with FBS alone as a means of comparing the different raw values obtained in each independent experiment.
Western immunoblotting analysis
Cells were trypsinized and centrifuged as described above, then plated at a density of 8 x 104 cells/well onto 24-well plates. After overnight incubation, cells were treated with medium containing 10% human serum (pregnant or nonpregnant) and antibiotics. After 24 h, cells were lysed and analyzed as described by Vidal-Puig et al. (38). In addition to the protease inhibitors described, we added 1 U/µL deoxyribonuclease I (Roche, no. 776 785). Protein lysates (50 µg) were mixed with Laemmli buffer and loaded onto a 10% SDS-PAGE gel with a 5% stacking gel. After electrophoresis, proteins were transferred to nitrocellulose using a Transblot apparatus (Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacturer’s instructions. The transfer time was 1 h, and 0.025% SDS was added to the standard buffer. After Ponceau S staining to verify equal protein transfer, blots were developed using standard procedures. Times and conditions of incubations: blocking, 1 h at room temperature; primary antibody (sc-7273, Santa Cruz Biotechnology, Inc.) 1:100 overnight at 4 C; and secondary antibody (sc-2005) 1:2000 for 45 min at room temperature. Blots were developed using ECL reagents (Amersham Pharmacia Biotech, Arlington Heights, IL) and were exposed to film.
Activation of PPRE by pregnancy serum
EP-JEG cells were plated as described above in 12-well plates.
After overnight incubation, cells were rinsed with PBS and treated in
duplicate with medium containing 10% human serum and antibiotics for
24 h as described in the text. Cells were lysed, and luciferase
activity was analyzed as described above (see treatment with activators
and inhibitors). As a control, some wells were also treated with FBS or
FBS plus 1 µmol/L 15dJ2 to verify normal
signaling. Again, after subtraction of signal observed with lysis
buffer alone, data were normalized to FBS controls. Pregnancy serum
(150 µL) was heated at 65 C for 20 min in a heat block, then placed
on ice for about 5 min. A second 150-µL aliquot was left at room
temperature (
22 C) for 6 h; a third aliquot was thawed
immediately before use. Each aliquot was added to 1.35 mL medium
(above) and used to treat triplicate wells of transfected JEG-3 cells
for 24 h. Luciferase activity was analyzed as described above for
JEG-3 cells.
Statistics
Results are presented as the mean ± SD. Because of limited sample size and non-Gaussian distributions, data were analyzed using nonparametric methods (Kruskal-Wallis tests for multiple comparisons and Mann-Whitney U tests for post-hoc and two-way analyses). Results were considered significant when two-tailed tests had values of P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is expressed in the normal placental bed
We used tissue sections from the placental bed to examine
expression of PPAR and - in pregnancy. Cytotrophoblasts were
identified in anchoring villi, decidua, and maternal spiral arteries by
cytokeratin staining (Fig. 1
, A, C, and
E, respectively). Although we were unable to detect expression of
PPAR in these experiments (data not shown), we observed expression
of PPAR in the nuclei of invasive cytotrophoblasts throughout the
placenta (Fig. 1, B, D, and F). In columns of the anchoring villi (Fig. 1, A and B), we observed expression of PPAR in cytokeratin-positive
invasive cytotrophoblasts. However, the noninvasive, differentiated
cells of the syncytiotrophoblast showed no expression of PPAR, nor
did maternal decidual or vascular cells.
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, C and D) also showed expression of PPAR. Finally,
cytotrophoblasts lining the maternal uterine spiral arteries (Fig.
1, E and F) showed intense nuclear expression of PPAR. We could
identify PPAR protein-expressing nuclei in about half the invasive
cytotrophoblasts observed throughout the placenta.
To determine whether PPAR expression also is detected in
vitro, we determined whether the PPAR protein is expressed in
cultured placental cells. Western immunoblot analysis of primary
cytotrophoblasts showed that PPAR protein expression is present in
freshly isolated trophoblasts (Fig. 2, lane 1) and after 24 h in culture (Fig. 2, lane 2). Analysis of
six independent preparations of primary cytotrophoblasts showed that
two of three first trimester samples and all three second trimester
samples expressed PPAR protein (data not shown).
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gene is expressed in cultured
placental and fetal endothelial cells. RT-PCR identified mRNA for
PPAR (Fig. 3) in primary
cytotrophoblast cells and in JEG-3 and JAr choriocarcinoma cell lines
(lanes 1, 3, and 6). In addition, HUVE cells expressed PPAR mRNA
(lane 9). Testis, a control tissue with no expression of PPAR (39),
showed no product in these studies (lane 12). Restriction enzyme
digestion with HincII generated the expected 108- and 76-bp
DNA fragments, verifying the specificity of the PCR products (lanes 2,
4, 7, and 10). Control reactions performed on the same RNA samples
without reverse transcriptase enzyme showed no PCR products (data not
shown).
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To examine the usefulness of JEG-3 cells as a model to study
the role of PPAR in pregnancy, we tested regulation of PPAR
activity. We used a previously described reporter gene construct (7)
that contains three consecutive PPAR response elements
(PPRE3) positioned upstream of a TK promoter and
firefly luciferase complementary DNA. JEG-3 cells were cotransfected
with this construct and a second control plasmid containing the
Renilla luciferase complementary DNA under control of a
constitutive TK promoter alone. Cells were then treated with the
PPAR-specific activator, 15dJ2. For
quantification of gene expression, firefly luciferase activity was
measured and normalized to Renilla luciferase activity. No
treatments resulted in detectable stimulation or inhibition of
Renilla luciferase activity (data not shown). As shown in
Fig. 4, transiently transfected JEG-3
cells showed a dosage response to 15dJ2, with an
EC50 of approximately 3.5 x
10-7 mol/L, similar to
other in vitro studies using this activator.
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As PPAR activation by 15dJ2 appeared to
be regulated normally in JEG-3 cells, we constructed a stably
transfected cell line (EP-JEG) to more reproducibly study
transcriptional activation by PPAR in placental cells. EP-JEG cells
responded to 15dJ2 and rosiglitazone with
EC50 values of approximately 3 x
10-7 and 1 x
10-7 mol/L, respectively
(Fig. 4; data for rosiglitazone not shown). This is consistent with the
responses we see in transiently transfected JEG-3 cells and is also
consistent with published values for other in vitro assay
systems (8, 9, 40).
JEG-3 and EP-JEG cells respond similarly to PPAR activators and
inhibitors
In addition to 15dJ2, rosiglitazone is a
highly specific PPAR activator (40), whereas the inflammatory
cytokine TNF is a physiological inhibitor of PPAR activation and
action (10, 11, 12, 13). We therefore examined the ability of all three
compounds to affect signaling via PPAR in our two cell lines. JEG-3
cells were transfected as before and then both JEG-3 and EP-JEG cells
were treated with 15dJ2 (1 µM),
rosiglitazone (1 µM), TNF (100 ng/mL), or a
combination of TNF and 15dJ2 in the above
concentrations. Figure 5 shows that JEG-3
and EP-JEG cells respond similarly and significantly (P
< 0.01, by Kruskal-Wallis test) to these compounds and in all respects
appear to show normal regulation (both activation and inhibition) of
PPAR pathways.
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expression and activation
As circulating levels of fatty acids and lipid metabolites
increase during pregnancy, and these factors are known to activate
PPAR (41, 42), we examined the effects of pooled sera from six
nonpregnant and six normal pregnant women on PPAR protein expression
in cultured JEG-3 cells. JEG-3 cells incubated with sera for 24 h
were lysed and analyzed for PPAR protein by Western immunoblot
analysis (Fig. 6). Cells treated with
sera from men (data not shown) or nonpregnant women (Fig. 6, lane 1)
showed little or no expression of PPAR protein. In contrast, cells
treated with sera from pregnant women (Fig. 6, lane 2) showed high
levels of PPAR expression. This result was observed in multiple
experiments using three independent pregnant serum pools. Similar
results were observed in cultured HUVE cells (data not shown).
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protein expression also reflected
increased activation, we treated EP-JEG cells with individual sera from
six pregnant and six nonpregnant women. As shown in Fig. 7, average luciferase activity was nearly
doubled in cells treated with pregnancy sera (1.95 ± 0.45-fold;
P < 0.005, by Mann-Whitney U test). Only one of the
six pregnancy samples failed to induce luciferase activity
significantly (1.75-fold or higher). In contrast, all six nonpregnant
samples showed luciferase activity similar to that of FBS alone
(0.80 ± 0.24-fold). It should be noted that JEG-3 cells displayed
similar signaling results (data not shown). Thus, we conclude that
treatment of cells with sera from pregnant women results in both
increased PPAR expression and increased activation of
PPAR-regulated genes.
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). This heat resistance suggests
that the factor(s) involved in the activity is not a protein. Indeed,
our data are consistent with an activator that is a prostanoid or other
fatty acid derivative, similar to natural PPAR ligands that have
been identified to date.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein is localized
in invasive human placental cytotrophoblast nuclei in vivo
and in cultured primary trophoblasts in vitro. We have also
shown that PPAR mRNA is abundant in cultured trophoblasts and HUVE
cells in vitro. We identified and characterized
choriocarcinoma cell lines (JEG-3 and EP-JEG) with endogenous
expression and apparently normal regulation of PPAR. Using these
cell lines, we show that circulating factors in pregnancy serum
up-regulate the expression and activation of PPAR in
vitro.
There are several reasons to believe that PPAR plays a role in
normal pregnancy. As both glucose and lipid metabolism are altered in
pregnancy, the role of PPAR as a common regulator of these systems
is particularly appealing (4). We found that PPAR is expressed in
extravillous cytotrophoblasts in vivo; however, we saw no
expression of PPAR in these tissues. Invasive cytotrophoblasts
express PPAR throughout the placental bed and into the maternal
spiral arteries. However, we found no expression of PPAR in the
syncytiotrophoblast, the terminally differentiated trophoblast progeny.
This finding suggests that PPAR may be required for the maintenance
of an invasive trophoblast phenotype, but not for the transport and
endocrine functions of syncytiotrophoblast. Further studies will
explore these questions.
We have identified a cell line (JEG-3) that is useful to study
the role of PPAR, particularly as it relates to pregnancy. The cell
line has native expression of PPAR, and this receptor appears to be
regulated normally in vitro. Stimulation of cells with
15dJ2 showed a dose-dependent response of the
PPRE3-luciferase construct with an
EC50 of about 3.5 x
10-7 mol/L, consistent
with the EC50 found in other cell systems.
PPAR signaling in these cells was inhibited by TNF, a cytokine
known to inhibit PPAR expression and action in other cells
(10, 11, 12, 13).
In addition, we created a stably transfected cell line from JEG-3
cells, EP-JEG, which provides a simple and reproducible way to assay
PPAR signaling in vitro. EP-JEG cells behave remarkably
similarly to transfected JEG-3 cells in all of our assays; their key
advantage is that they do not require transfection of a reporter
construct to assay PPAR activation.
Using these two cell lines, we established that normal human pregnancy
serum contains factors that increase the expression and activation of
PPAR. Such factors are not present in nonpregnant serum. Expression
of PPAR protein was dramatically increased from undetectable to high
levels of expression in a Western immunoblot assay. PPRE-dependent
luciferase signaling was increased by 1.95 ± 0.45-fold in cells
treated with pregnancy serum compared to that in cells treated with
nonpregnant serum. These levels of signaling are similar to those in
cells incubated with FBS in the presence or absence of 1 µmol/L
15dJ2, respectively.
The presence of PPAR activators in pregnancy serum supports a role
for PPAR action in pregnancy. The dramatic alterations in glucose
and lipid metabolism seen during pregnancy certainly are consistent
with changes induced by a master regulator such as PPAR. Lim
et al. identified a potential role for PPAR in
trophoblast implantation of mice (15). Additionally, Bastie et
al. Showed that exogenous expression of PPAR may lead to
induction of PPAR expression in vitro (43). Taken
together, these findings point to the possibility that the human
PPAR homolog NUC1 along with PPAR are important in implantation
and human pregnancy. Recent work by Barak et al. identifies
PPAR as necessary for placental development in mice (14). Thus, it
is possible that NUC1 and PPAR both play important roles in the
development and homeostatic maintenance of the placenta throughout
pregnancy.
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Acknowledgments |
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Footnotes |
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Received March 10, 2000.
Revised June 9, 2000.
Accepted June 28, 2000.
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References |
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A. Tarrade, K. Schoonjans, L. Pavan, J. Auwerx, C. Rochette-Egly, D. Evain-Brion, and T. Fournier PPAR{gamma}/RXR{alpha} Heterodimers Control Human Trophoblast Invasion J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 5017 - 5024. [Abstract] [Full Text] [PDF]
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 A. Tarrade, K. Schoonjans, J. Guibourdenche, J. M. Bidart, M. Vidaud, J. Auwerx, C. Rochette-Egly, and D. Evain-Brion PPAR{gamma}/RXR{alpha} Heterodimers Are Involved in Human CG{beta} Synthesis and Human Trophoblast Differentiation Endocrinology, October 1, 2001; 142(10): 4504 - 4514. [Abstract] [Full Text] [PDF]
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E. C. Person, L. L. Waite, R. N. Taylor, and T. S. Scanlan Albumin Regulates Induction of Peroxisome Proliferator-Activated Receptor-{{gamma}} (PPAR{{gamma}}) by 15-Deoxy-{{Delta}}12-14-Prostaglandin J2 in Vitro and May Be an Important Regulator of PPAR{{gamma}} Function in Vivo Endocrinology, February 1, 2001; 142(2): 551 - 556. [Abstract] [Full Text] [PDF]
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) and EP-JEG cells (•) treated with 15dJ2.
Cells were prepared as described in Materials and
Methods and treated in triplicate with the indicated
concentrations of 15dJ2 for 24 h. Fold stimulation
relative to the effect of treatment with 10% FBS alone is shown. The
mean ± SD from representative experiments are shown
for each cell type. Similar results were found in triplicate
determinations of at least two independent experiments.
) and EP-JEG (
) cells. Cells were treated as described
in Materials and Methods. Luciferase activation
normalized to 10% FBS treatment alone is shown. Values represent the
mean ± SD for combined normalized data from
independent experiments. Concentrations of compounds:
15dJ2, 1 µmol/L; rosiglitazone, 1 µmol/L; and TNF
