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and Prostaglandin E2
From the Center for Research on Reproduction and Women’sHealth,* University of Pennsylvania Medical Center,Philadelphia, Pennsylvania; and the Department ofBiochemistry, MRC Immunochemistry Unit,University of Oxford, Oxford, United Kingdom
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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(TNF-), an established inducer of TSG-6. TNF- stimulated
TSG-6 mRNA accumulation in a dose- and time-dependent manner,
with the maximal response observed at 10 ng/ml after 6 hours of
incubation. PGE2 stimulated TSG-6 mRNA expression,
but the magnitude of response was substantially less than that produced
by TNF-, and it was maximal only after 24 hours of
incubation. Quantitative real-time polymerase chain reaction was
performed to assess the induction of TSG-6 mRNA and nascent transcripts
at 24 hours of treatment. Induction of TSG-6 mRNA and nascent
transcripts in response to 10 µmol/L of PGE2 was 5.7-fold
and 6.3-fold greater than control values, respectively,
whereas TNF- (10 ng/ml) induced TSG-6 mRNA and nascent transcripts
by 80-fold and 134-fold, respectively. TNF- and
PGE2 stimulated secretion of TSG-6 into the culture medium
as detected by Western blotting. The effects of PGE2 on
secretion of TSG-6 were delayed compared to TNF-. A 1.3-kb fragment
of the human TSG-6 proximal promoter drove
luciferase expression in transfected hCSMCs. PGE2
increased TSG-6 promoter activity 1.75-fold.
Paradoxically, TNF- reduced TSG-6 promoter
activity by 50%. We conclude that hCSMCs express the hyaladherin
TSG-6; that TSG-6 expression in these cells is regulated by
PGE2 as well as proinflammatory cytokines; responses of
hCSMCs to TNF- and PGE2 are distinct in terms of
magnitude and the time course; and PGE2 and TNF- exert
different effects on the TSG-6 proximal
promoter.
In the present study we examined the expression of TSG-6 by human cervical smooth muscle cells (hCSMCs) in culture, focusing on the effects of molecules that are known to promote cervical ripening, proinflammatory cytokines, and prostaglandin E2 (PGE2).
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Proliferating human cervical smooth muscle cells (hCSMCs) (Clonetics, San Diego, CA) that we have previously demonstrated to retain the molecular phenotype of smooth muscle cells,18 were grown in Smooth Muscle Cell Growth Medium-2 basal medium (Clonetics) supplemented with 5% fetal bovine serum. This medium contains human epidermal growth factor (0.5 ng/ml), human fibroblast growth factor (1.0 ng/ml), insulin (5 µg/ml), gentamicin (50 µg/ml), and amphotericin B (50 µg/ml). hCSMCs were cultured at 37°C under an atmosphere of 5% CO2 in air. Subcultures of hCSMCs from passages 3 to 6 were used in all of the experiments. Each experiment was reproduced on at least two occasions with similar results.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and TSG-6 Probe Preparation
Five µg of total RNA extracted from hCSMCs was reverse-transcribed to produce cDNA using reverse transcriptase and oligo dT (Promega, Madison, WI) as a primer. The TSG-6 cDNA was amplified using 10% of the RT reaction in 100 µl containing 50 pmol of forward primer, 50 pmol of reverse primer, 5 U of Taq polymerase (Life Technologies, Inc., Grand Island, NY) with 0.2 mmol/L of dNTPs and 1.5 mmol/L of MgCl2. The sequences of the primers used to generate the TSG-6 probe were: forward primer 5'-ATTTGTGAGCAGCCCCTAAC-3' and reverse primer 5'-AGTGAGATCAAAGGAGTTCC-3'. The expected size of the PCR product is 951 bp. PCR was performed in a 9600 GeneAmp PCR thermal cycler using the following conditions: 94°C (1 minute) for one cycle, 94°C (1 minute), 57°C (1 minute), 72°C (1.5 minutes) for 35 cycles, and a final incubation at 72°C for 7 minutes. The PCR product was subcloned into PCR 2.1 vector (Invitrogen, Carlsbad, CA) and sequenced to verify that it encoded the TSG-6 cDNA. To obtain the insert for Northern blotting, the plasmid containing the sequence-verified PCR product was digested with EcoRI and the insert was purified using a gel extraction kit (Qiagen, Valencia, CA) before labeling by the random primer method.
RNA Isolation and Northern Blot Analysis
Subconfluent cultures of hCSMCs were grown in medium
supplemented with 1% fetal bovine serum for 24 hours before treatment
with tumor necrosis factor (TNF)- (0.01 to 20 ng/ml) (R&D System,
Minneapolis, MN) or PGE2 (5 to 40 µmol/L)
(Sigma Chemical Co., St. Louis, MO) for up to 72 hours. Total RNA was
extracted from the cultures with Trizol reagent (Life Technologies,
Inc.) using procedures recommended by the manufacturer. Equal
amounts of RNA (30 to 50 µg/lane) were separated on 1%
agarose-formaldehyde-denaturing gels and transferred to nylon
membranes. Membranes were hybridized with
32P-labeled TSG-6 cDNA at 42°C for 16 to 18
hours, followed by two sequential washings for 15 minutes in 2x sodium
chloride/sodium phosphate ethylenediaminetetraacetic acid (SSPE), 0.1%
sodium dodecyl sulfate (SDS) at 37°C, and two washings in 0.1x SSPE,
0.1% SDS at 55°C. Blots were analyzed using a phosphoimager and then
exposed to Kodak X-Omat AR film (Eastman-Kodak, Rochester, NY). The
relative abundance of TSG-6 mRNA was normalized to 28S rRNA.
Effects of Actinomycin D (Act D) and Cycloheximide (CHX) on
TNF-- and PGE2-Induced mRNA Expression
Subconfluent hCSMCs were cultured with 2 µg/ml of Act D or 50
µg/ml of CHX with 10 ng/ml of TNF- or 10 µmol/L of
PGE2 for 6 or 24 hours. Total RNA was subjected
to Northern blotting as described above.
Western Blot Analysis
hCSMCs were cultured in serum-free medium for 24 hours, and then
treated with TNF- (10 ng/ml) for 24 hours or
PGE2 (5 to 40 µmol/L) for up to 72 hours. The
conditioned medium was subjected to 10% SDS-polyacrylamide gel
electrophoresis and then Western blotting. The polyclonal antiserum
used in Western blotting was raised as described in Carrette and
colleagues15
except that a 17-amino acid peptide
(CTSTGNKNFLAGRFSHL) corresponding to the final 16-amino acid residues
of human TSG-619
and a nonauthentic N-terminal Cys residue
was used as the antigen. Supersignal West Femto Maximum Sensitivity
Substrate (Pierce, Rockford, IL) was used to detect antibody bound to
antigen.
TSG-6 Promoter Analysis
The promoter region of the human TSG-6 gene was amplified by PCR using a forward primer spanning bp -1320 to -1301 with a KpnI linker (5'-CGAGGTACCTCACTAACCCTATCTGTGAA-3') and a reverse primer spanning bp -20 to -1 with a NheI linker (3'-CTACACCTTTGGTCTACAAACGATCGAGC-5'). The 1.32-kb fragment spanning bp -1320 to -1 of the human TSG-6 gene was cloned into the pGL3 basic vector (Promega), which contains firefly luciferase as a reporter gene. 5'-deletion constructs were prepared by the subcloning of PCR products generated using various forward TSG-6-specific primers with attached linkers and the reverse primer described above. The forward primer used to generate the -756 TSG-6 promoter fragment spanned from bp -756 to -737 with a KpnI linker (5'-CGAGGTACCCCTTGATCGTCTTCTTCAAA-3'); the forward primer for the -160 TSG-6 promoter fragment spanned from bp -160 to -141 with a KpnI linker (5'-CGCGGTACCATTCTATCTCCTTAGTTTTG-3'); the forward primer for the -100 TSG-6 promoter fragment spanned from bp -100 to -81 with a KpnI linker (5'-CGCGGTACCTGAGATAATTGTGTAACTCT-3'). The sequences of all of these PCR-generated promoter fragments were confirmed to be identical to the corresponding TSG-6 promoter sequence previously reported by Lee and colleagues20 with the following exceptions that insertions of A, G, C, T, A, C, and TG between nucleotides -1292 and -1291, -867 and -866, -681 and -680, -678 and -677, -631 and -630, -472 and -471, and -53 and -52, respectively, were consistently found. These same sequence variations were identified independently in Dr. Day’s laboratory and have been deposited in EMBL with accession codes of AJ413948 and AJ413949.
Transfection and Cell Stimulation
hCSMCs (1 x 105) were seeded in
each well of 12-well plastic culture plates in medium supplemented with
1% fetal bovine serum. Cells were transfected using FuGENE 6
Transfection Reagent (Roche, Indianapolis, IN) with 0.5 µg/ml of each
of the promoter constructs, 0.5 µg/ml of the CMV-ß galactosidase
plasmid, and 2 µl/ml of FuGENE. After 24 hours, hCSMCs were incubated
in 0.1% ethanol (control), 10 ng/ml TNF-, or 10 µmol/L
PGE2 in medium supplemented with 1% fetal bovine
serum for 24 hours to determine the effect on TSG-6 promoter activity.
At the end of the culture period, cells were harvested and the cell
lysates were assayed for luciferase and ß-galactosidase activity as
described below.
Luciferase and ß-Galactosidase Assay
Luciferase activity was determined in a LUMAT LB 9507 luminometer (EG&G Berthold, Gaithersburg, MD) with Promega luciferin as substrate. ß-galactosidase activity was determined by a standard colorimetric assay using 2-nitrophenyl ß-D-galactopyranoside as substrate. The luciferase assay results were normalized to ß-galactosidase activity to correct for variations in transfection efficiency. Each treatment group contained triplicate cultures and each experiment was repeated three to four times. Relative luciferase units (RLU) defined as luciferase light units/ß-galactosidase activity are presented as means ± SE.
Reverse Transcription Reaction and Quantitative Real-Time PCR for TSG-6 mRNA
Proliferating hCSMCs were incubated with 0.1% ethanol vehicle or 10 µmol/L of PGE2 for 24 hours. Total RNA was extracted using Trizol reagent (Life Technologies, Inc.). To limit the possibility of detection of genomic DNA, total RNA was treated with RQ1-RNase-free DNase (Promega) for 30 minutes at 37°C before reverse transcription with Moloney murine leukemia virus-reverse transcriptase (Promega) as described by the manufacturer. Five µg of total RNA was reverse-transcribed to single-strand cDNA using 1 µl of oligo(dT)15 primer (Promega), 0.5 µl of RNase inhibitor from human placenta (Roche Molecular Biochemicals), and 1 µl of Moloney murine leukemia virus-reverse transcriptase (Promega) as described by the manufacturer.
Quantitative real-time PCR was performed to assess the induction of TSG-6 mRNA in hCSMCs in response to 10 µmol/L of PGE2. Primers for the analysis of the human TSG-6 gene elements were designed in the third exon: forward, 5'-TCATGTCTGTGCTGCTGGATG-3'; and reverse, 5'-GGGCCCTGGCTTCACAA-3'. The resulting cDNA was diluted 10-fold in sterile water and aliquots were subjected to quantitative real-time PCR. The real-time PCR reaction used 90 nmol/L of each primer and 12.5 µl of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Agarose gel electrophoresis indicated the presence of a 67-bp single band for the TSG-6 PCR product. To account for differences in starting material, the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers and probe reagents from Applied Biosystems were used as described by the manufacturer. The target and GAPDH PCR reactions were performed in separate tubes in triplicate and the average threshold cycle for the triplicate was used in all subsequent calculations.
Quantitative Real-Time PCR of TSG-6 Nascent Transcripts
To limit the possibility of detection of genomic DNA, 5 µg of total RNA was treated with RQ1-RNase-free DNase (Promega) as described above. For the human TSG-6 and human GAPDH nascent transcript expression, primers for reverse transcription were designed in the third and fourth intron of each gene, respectively; TSG-6: 5'-CAACCTCTTAGCAGCATGGAACTGT-3', GAPDH: 5'-TAGTTGCCTCCCCAAAGCAC-3'. Five µg of total RNA was reverse-transcribed to single-stranded cDNA as described above. The resulting amplicons derived from the specific TSG-6 and GAPDH intronic primers were diluted 10-fold in sterile water and aliquots were subjected to quantitative real-time PCR. The forward and reverse PCR primers for TSG-6 nascent RNA expression were the same as those described above for TSG-6 mRNA expression. For GAPDH nascent transcript expression, PCR primers were designed in the third exon (forward, 5'-GATTCCACCCATGGCAAATT-3'; reverse, 5'-AAGATGGTGATGGGATTTCCATT-3'). Optimization studies for detection of the nascent GAPDH transcript indicated that 90 nmol/L of each primer should be used in each 25-µl reaction. Agarose gel electrophoresis revealed the presence of a 83-bp single band for the nascent GAPDH transcript PCR product. PCR reactions for nascent TSG-6 and GAPDH transcripts were performed in separate tubes in triplicate and the average threshold cycle for the triplicates was used in all subsequent calculations.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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and PGE2 Increase TSG-6 Gene Expression by
hCSMCs
TNF- is an established inducer of TSG-6 expression. We first
defined the response of the hCSMCs to this proinflammatory cytokine.
TNF- had a dose-dependent stimulatory effect on expression of the
18S TSG-6 mRNA (Figure 1)
. As little as
0.1 ng/ml of TNF- raised TSG-6 message abundance, and a maximal
response was observed with 10 ng/ml. Time-course studies conducted with
10 ng/ml of TNF- (Figure 2)
revealed a
rapid increase in TSG-6 mRNA, detectable after 0.5 hours of treatment,
the first time point we examined, and reaching a maximum at 6 hours,
with mRNA levels declining modestly thereafter.
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that may
reflect desensitization or cell demise. In contrast to TNF-, which
rapidly induced TSG-6 mRNA, the response of hCSMCs to
PGE2 was more sluggish. A modest response was
seen at 6 hours and a peak in TSG-6 mRNA was observed at 24 hours of
treatment (Figure 4)
. Notably, TSG-6 mRNA
induction by PGE2 after 24 hours of treatment was
substantially lower than that observed with 10 ng/ml of TNF- based
on the signal strength of simultaneously hybridized blots and blots
containing RNA from TNF-- and PGE2-treated
cells.
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We performed quantitative real-time PCR to assess the induction of
TSG-6 mRNA and nascent transcripts, the latter being an index of
TSG-6 gene transcription, in hCSMCs treated with 10 µmol/L
of PGE2 for 24 hours (Table 1)
. The increase in TSG-6 mRNA and
nascent transcripts in response to 10 µmol/L of
PGE2 was 5.7-fold and 6.3-fold compared to the
0.1% ethanol control group, respectively. In simultaneously conducted
experiments TNF- treatment increased TSG-6 mRNA and nascent
transcript levels by 
80-fold and 134-fold, respectively. These
observations are consistent with the significant differences in TSG-6
induction in response to the prostanoid and the cytokine demonstrated
by Northern blotting. We also examined the effects of 8-Br-cAMP because
PGE2 is thought to act on hCSMCs via the
EP4 receptor, which is coupled to adenylate
cyclase.21
The response to 8-Br-Br-cAMP was
basically similar to that produced by PGE2.
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at either low concentrations (0.1 ng/ml) or concentrations that
maximally stimulate TSG-6 expression (10 ng/ml). Cells were treated
with each agent alone or in combination for 24 hours. We found no
evidence for PGE2 augmentation of the TSG-6 mRNA response
to TNF- at either low or high TNF- concentrations (data not
shown).
TNF- and PGE2 Stimulate TSG-6 Secretion, But with
Different Temporal Patterns
Exposure of hCSMCs to TNF- at 10 ng/ml for 24 hours resulted in
the release of the 39-kd TSG-6 protein into the culture medium as
detected by Western blotting (Figure 5)
.
In contrast, there was no detectable release of TSG-6 by hCSMCs exposed
to 10 µmol/L of PGE2 for 24 hours. However,
when hCSMCs were cultured with 10 µmol/L of
PGE2 for up to 72 hours, detectable TSG-6 was
present in the conditioned medium by 48 hours of incubation (Figure 6)
. The effects of
PGE2 on secretion of TSG-6 after 72 hours of
culture were dose-dependent with maximal secretion observed with 10 and
20 µmol/L of PGE2, and as with TSG-6 mRNA
expression, lower production of TSG-6 was observed with concentrations
of 30 and 40 µmol/L (Figure 7)
.
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and PGE2
Requires On-Going RNA But Not Protein Synthesis
The TNF-- and PGE2-stimulated rise in
TSG-6 mRNA was not dependent on on-going protein synthesis as it
occurred in the presence of the inhibitor, CHX that blocks de
novo protein synthesis by >95% at the concentration used in this
study (50 µg/ml) (Figure 8)
. In fact,
CHX alone caused a modest increase in TSG-6 mRNA (2.4-fold greater than
control, normalized to 28S rRNA) and augmented the effects of
PGE2 (4.3-fold at 6 hours of treatment and
14.67-fold at 24 hours), but not those of TNF-. However, TNF- and
PGE2-stimulated TSG-6 mRNA expression required
on-going RNA synthesis as Act D completely blocked the rise in TSG-6
mRNA in both TNF-- and PGE2-treated hCSMCs
without affecting basal levels at 6 hours of treatment and reducing
basal mRNA abundance normalized to 28S rRNA by 60% after 24 hours
of treatment.
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To examine the regulation of TSG-6 gene transcription,
we cloned the TSG-6 promoter from hCSMC genomic DNA. A
1.3-kb fragment of the human TSG-6 proximal promoter drove
luciferase expression in transfected hCSMCs. Treatment of the
cells with 10 µmol/L of PGE2 after 24 hours of
incubation increased TSG-6 promoter activity 1.75-fold
(Table 2)
. Paradoxically, TNF- (10
ng/ml) reduced TSG-6 promoter activity by 64%, indicating
that PGE2 and TNF- regulate TSG-6
transcription by different mechanisms. We also examined four
5'-deletion constructs (Figure 9)
. Ten
µmol/L of PGE2 increased the activity of the
-756 and -160 constructs, but not the -100 construct that had only
modest promoter activity compared to the empty pGL3 vector. Notably
removal of sequences between positions -1320 bp and -756 bp tended to
enhance promoter function. A similar result has been reported by Lee
and colleagues20
who studied TSG-6 promoter activity in
human FS-4 foreskin fibroblasts.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Although the functions of HA in the cervix during parturition are
becoming clear, there has been little attention given to the HA-binding
proteins that are thought to mediate and/or modulate the actions of HA.
TSG-6 is one of the HA-binding proteins containing a single link module
composed of two -helices and two triple-stranded ß-sheets arranged
around a large hydrophobic core.11
TSG-6 interacts with HA
via its link module and amino acid residues involved in ligand binding
have recently been determined.17,29
TSG-6 has been
implicated in the regulation of leukocyte migration and its pattern of
expression suggests that it may be involved in extracellular matrix
remodeling.16,30
Elevated TSG-6 protein levels have been
found in the synovial fluid of arthritic patients,31
and
recombinant TSG-6 has potent anti-inflammatory activity in
vivo.16
Based on these results, it was suggested that
TSG-6 constitutes part of a cytokine-initiated feedback loop that
operates to down-regulate the inflammatory response.16
TSG-6 has been shown to form a stable, probably covalent, 120-kd
complex with the serine protease inhibitor, inter- inhibitor
(II).32
It has been reported that this complex has
significantly increased anti-plasmin activity over II
alone.16
Thus TSG-6 may be important in regulating
leukocyte migration and matrix remodeling because plasmin enhances the
activation of latent metalloproteinases involved in extracellular
matrix breakdown.13,16,33
Our observations demonstrate that hCSMCs express TSG-6, and that TSG-6
expression in these cells is regulated, as expected, by proinflammatory
cytokines. However, we have also established for the first time that
PGE2 controls the TSG-6 gene. Notably,
the responses of hCSMCs to TNF- and PGE2 are
distinct in terms of both magnitude and time course. We found that
TNF- and PGE2 treatment of hCSMCs increased
nascent TSG-6 transcripts demonstrating that the rise in TSG-6 mRNA is
at least in part the result of increased TSG-6
transcription. This observation is consonant with the fact that Act D
blocked the induction of TSG-6 mRNA. Yet, the effect of
PGE2 and TNF- on TSG-6 proximal
promoter activity suggest that the regulatory elements responsible for
increased transcription are different. This is not unanticipated
because PGE2 acts on cervical fibroblasts through
the EP4 receptor that couples to adenylate
cyclase,21
whereas proinflammatory cytokines activate
receptors that do not immediately engage the cAMP signaling cascade.
The ability of cAMP-mediated signaling pathways to influence TSG-6
expression was verified by demonstrating that 8-Br-cAMP (1 mmol/L for
24 hours) increased TSG-6 mRNA and nascent transcript accumulation. The
modest increase in TSG-6 mRNA abundance in CHX-treated cells and the
augmentation of the response to PGE2 may suggest
an effect of CHX on TSG-6 mRNA stability or possibly inhibition of
synthesis of a transcriptional silencer. The fact that CHX did not
block the effects of PGE2 on TSG-6 gene
expression argues against the involvement of a prostanoid-induced
protein autocrine factor in the TSG-6 response.
Lee and colleagues20
reported that sequences between -165
to +78 in the TSG-6 promoter confer regulation by
proinflammatory cytokines on this gene. This region contains a
potential binding site for AP-1 as well as other transcription factors.
However, the transcriptional response of the TSG-6 promoter
construct reported by Lee and colleagues20
in FS-4 cells
to TNF- treatment was modest at best compared to the response to
interleukin-1. The magnitude of stimulated promoter activity in the
study of Lee and colleagues20
also did not correspond to
the change in TSG-6 mRNA or the results of nuclear run-on assays. Our
findings suggest that elements in the TSG-6 gene outside of
those included in our promoter constructs and those of Lee and
colleagues20
must contribute to the transcriptional
response to TNF- as well as the maximal response to
PGE2.
The production of TSG-6 in the cervix driven by proinflammatory cytokines and PGE2, molecules that promote cervical ripening, implicates TSG-6 in the cervical ripening process. Based on existing information, we speculate that TSG-6 modulates the inflammation-like biochemical and cellular changes in the cervix by retaining leukocytes in the cervix while at the same time exerting anti-inflammatory actions and restricting proteolytic activity so that alterations in the extracellular matrix are spatially and temporally regulated.
TSG-6 can be detected in human cervical fluid of women who present with symptoms of preterm labor (T Fujimoto and JF Strauss, unpublished observations). This finding is consistent with the notion that the cervix produces TSG-6 in situ. It also raises the possibility that detection of TSG-6 could serve as marker for impending cervical changes leading to preterm birth.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grant HD34612) and the Bill and Melinda Gates Foundation.
Accepted for publication January 2, 2002.
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References |
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, IL-1ß, and LPS-inducible secreted glycoprotein associated with arthritis. J Immunol 1993, 151:6593-6601[Abstract]
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