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Regular Articles |
From the Center for Research on Reproduction and Women's
Health*
and the Department of
Urology,
University of Pennsylvania Medical
Center, Philadelphia, Pennsylvania, and the Division of Pulmonary and
Critical Care Medicine,§
Brigham and Women's
Hospital and Harvard Medical School, Boston, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) and examined expression of the
elastinolytic enzyme, cathepsin S, the collagen
metabolizing matrix metalloproteinases (MMP)-1, -3,
-9, and the tissue inhibitor of metalloproteinase (TIMP)-1 and
-2. A time course analysis revealed that 10 ng/ml of TNF- induced
cathepsin S, MMP-1, -3, and -9 mRNA expression
with the maximal response observed after 24–48 hours. TNF- induced
cathepsin S, MMP-1, -3, and -9 mRNA expression
in a dose-dependent manner: the maximal effect was observed at a
concentration of 10 ng/ml, with appreciable increases observed
at concentrations of 0.1 to 1.0 ng/ml. In contrast, TIMP-1 and
-2 mRNAs were not significantly increased by TNF- treatment.
Interleukin-1ß produced a pattern of gene expression in the CSMC
similar to that observed following TNF- treatment. Western blot
analysis and zymography confirmed the induction of proMMP-1,
-3, and -9 in response to TNF-, but MMP-2
immunoreactivity and zymographic activity were unaffected. TNF-
increased secretion of procathepsin S, but did not affect
TIMP-1 and reduced TIMP-2 production. We conclude that CSMC are targets
of pro-inflammatory cytokines, which induce a repertoire of
enzymes capable of degrading the cervical extracellular matrix. The
induction of these enzymes may facilitate the normal ripening of the
cervix at term and participate in the premature cervical changes
associated with preterm labor.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The restructuring of the cervical extracellular matrix has been
attributed to the release of proteases from invading white blood cells
and from cervical fibroblasts.4-7
Pro-inflammatory
cytokines are believed to participate in this process.8,9
The cellular targets of these cytokines may include other resident
cells in the cervix, because interleukin (IL)-1 has been reported to
increase the production of an elastase-like enzyme from human cervical
fibroblasts10
as well as matrix metalloproteinases
(MMPs).11
There are, however, no reported studies on
matrix-metabolizing enzyme expression by human cervical smooth muscle
cells (CSMC).
Several enzymes could potentially be involved in the extracellular matrix remodeling associated with cervical ripening, including the MMPs that degrade fibrillar collagen, MMP-1 and MMP-13, which are produced by mesenchymal as well as some epithelial cells, and leukocyte collagenase, MMP-8.12 Leukocytes also produce a distinct elastase.13 Cathepsins, lysosomal cysteine proteinases, also display elastase activity. Among the cathepsins, cathepsin S is a potential candidate enzyme involved in cervical elastin metabolism because of its potent elastinolytic activity at neutral pH.14 Elastin can also be broken down by MMP-9, an enzyme produced by various cell types including leukocytes. In addition, MMP-2 and MMP-9 hydrolyze collagen fragments produced by the action of the interstitial collagenases.12
In the experiments reported here, we tested the hypothesis that
pro-inflammatory cytokines regulate the expression of matrix-degrading
enzymes by CSMC. We report that tumor necrosis factor- (TNF-) and
IL-1ß stimulate the expression of matrix-degrading MMPs and cathepsin
S, suggesting that release of protease from CSMC plays a role in the
normal process of cervical ripening as well as the premature changes in
cervical structure associated with preterm labor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human CSMC isolated from uteri of nonpregnant women removed for
benign disease were purchased from Clonetics (San Diego, CA). The CSMC
were grown in Smooth Muscle Cell Growth Medium-2 basal medium
supplemented with 5% fetal bovine serum (Clonetics). 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) at 37°C under an atmosphere
of 5% CO2 in air. Subcultures of CSMC from
passages 3–7 were used in all of the experiments. Before each
experiment, CSMC were cultured in medium supplemented with 1% fetal
bovine serum or serum-free medium for 24 hours. CSMC were
cultured for the indicated times with or without recombinant human
TNF- or IL-1ß (R & D Systems, Minneapolis, MN).
RNA Isolation and Northern Blot Analysis
Subconfluent cultures of CSMC were grown in medium supplemented
with 1% fetal bovine serum for 24 hours before treatment with TNF-
(0.01–20 ng/ml) or IL-1ß (10 ng/ml) for up to 48 hours. Total RNA
was extracted from the cultures with Trizol reagent (Gibco-BRL, Grand
Island, NY) using procedures recommended by the manufacturer. Equal
amounts of RNA (40 µg/lane) were separated on 1%
agarose-formaldehyde denaturing gels, transferred to nylon membranes,
and hybridized sequentially with 32P-labeled
cathepsin S, MMP-1, MMP-3, MMP-9, and TIMP-1 and TIMP-2 cDNAs at 42°C
for 16–18 hours, followed by two sequential washings for 10 minutes in
2X 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 exposed to Kodak X-Omat
AR film and then analyzed with a phosphoimager (Molecular Dynamics,
Sunnyvale, CA) for quantitation. The relative abundance of mRNAs was
normalized to 28S rRNA.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA extracted from human WISH cells was used for RT-PCR to
generate the cathepsin S cDNA probe and total RNA extracted from CSMC
was used to generate the MMP-1, -3, -9, and TIMP-1 and -2 cDNAs. RNA
was reverse transcribed to produce cDNA using reverse transcriptase
(Promega, Madison, WI) and oligo dT as a primer. The cDNAs for
cathepsin S, MMP-1, -3, -9, and TIMP-1 and -2 were amplified using 10%
of the RT reaction in 100 µl containing 50 pmol forward primer, 50
pmol reverse primer, and 5 U Taq polymerase (Perkin-Elmer,
Foster City, CA), with 0.2 mmol/L dNTPs and 1.5 mmol/L
MgCl2. The sequences of the synthesized primers
and the expected sizes of the PCR products are shown in Table 1
. PCR was performed in a 9600 GeneAmp
PCR thermal cycler using the following conditions: for cathepsin S and
MMP-9, 94°C (1 minute) for 1 cycle, 94°C (1 minute), 57°C (1
minute), 72°C (1 minute) for 30 cycles, and the final incubation at
72°C for 7 minutes; for MMP-1, 94°C (1 minute) for 1 cycle, 94°C
(1 minute) 63°C (1 minute), 72°C (2 minutes) for 30 cycles, and
final incubation at 72°C for 7 minutes; for MMP-3, 94°C (1 minute),
57°C (1 minute), 72°C (2 minutes) for 30 cycles, and final
incubation at 72°C for 7 minutes; for TIMP-1 and -2, 94°C (1
minute), 57°C (1 minute), 72°C (1 minute) for 30 cycles, and final
incubation at 72°C for 7 minutes. The PCR products were ligated into
either PCR 3.1 or PCR 2.1 vector (Invitrogen, Carlsbad, CA), and
the sequences were confirmed. To obtain the inserts for Northern
blotting, the plasmids were digested with BamHI and
KpnI (cathepsin S) or EcoRI (MMPs and TIMPs) and
the inserts were purified using a gel extraction kit (Qiagen, Stanford,
CA) before labeling by the random prime method.
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-smooth muscle actin
cDNA. Western Blot Analysis
Cathepsin S, MMP-1, -2, -3, and TIMP-1 and -2 in conditioned
medium were analyzed by Western blotting.15
CSMC were
cultured in serum-free medium for 24 hours, and then treated with
TNF- (10 ng/ml) for 48 hours. The conditioned medium, normalized to
equal numbers of cells for each treatment group, was collected and
concentrated under vacuum at -50°C. The samples were subjected to
Western blotting using a rabbit polyclonal antibody raised against
human cathepsin S,16
and mouse monoclonal antibodies
raised against human MMP-1, -2, -3, and TIMP-1 and -2 (Calbiochem, La
Jolla, CA). The Amersham enhanced
chemiluminescence system (Amersham Life Sciences, Arlington
Heights, IL) was used to detect antibody bound to antigen.
Zymography
CSMC were cultured in serum-free medium for 24 hours before
treatment with TNF- (10 ng/ml). The conditioned medium was collected
after 48 hours of treatment and concentrated under vacuum at -50°C.
The aliquots normalized to equal numbers of cells were then subjected
to SDS polyacrylamide gel electrophoresis in 7.5% or 10%
polyacrylamide gels containing 1 mg/ml gelatin or casein under
nonreducing conditions.17
After washing the gels in 2.5%
Triton-X 100 (15 minutes x 2) to remove the SDS, the gels were
incubated in a buffer containing 50 mmol/L Tris-HCl, pH 7.4, 30 mmol/L
CaCl2, 150 mmol/L NaCl at 37°C for 16–18
hours. The gels were then stained with Coomassie Brilliant Blue G250
and destained in a solution consisting of 10% methanol, 10% acetic
acid, and 10% glycerol.
Immunocytochemistry
The presence of smooth muscle -actin in the CSMC was detected
by immunocytochemistry with a monoclonal antibody and immunoperoxidase
technique using reagents purchased from Sigma Chemical Co. (St. Louis,
MO). Immunostaining was carried out according to the supplier's
protocol. Controls included incubations in the absence of the primary
antibody.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CSMC grew as spindle-shaped cells in culture. Virtually all of
these cells stained positively for the smooth muscle cell marker,
smooth muscle -actin. (Figure 1)
. We
also carried out an RT-PCR analysis for smooth muscle cell-specific
transcripts encoding myosin heavy chains and 17-kd light chains (Figure 2)
.18
Using primers to
specifically amplify transcripts for SM1 and SM2, myosin heavy chain
isoforms that are different at the C-terminal region,19,20
we found that the CSMC expressed predominantly SM1. The analysis of
myosin light chain transcripts indicated that the cultured CSMC had a
light chain pattern consisting of 74% LC17b, which is smooth
muscle-specific, and 26% of the LC17a isoform, which is expressed by
both smooth muscle and non-muscle cells in humans.21
The
primers for actin amplified human smooth muscle -actin cDNA.
Collectively, these observations that the CSMC express transcripts
characteristic of smooth muscle20,22
document that the
cells studied were members of the smooth muscle lineage.
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Increased the Expression of MMP-1, -3, -9, and Cathepsin S
but not TIMP-1 and -2 mRNAs in CSMC
TNF- at a dose of 10 ng/ml increased the steady state levels of
MMP-1, -3 and -9 and cathepsin S mRNAs over a 48-hour incubation
(Figure 3)
. Significant increases in the
transcripts were seen within 6 hours after addition of the cytokine. In
contrast, transcripts encoding TIMP-1 and TIMP-2 were only modestly
affected. The effects of TNF- on mRNA expression were dose-dependent
(Figure 4)
. An increase in MMP-3 mRNA was
evident at a TNF- concentration of 0.1 ng/ml. Increases in MMP-1 and
-9 and cathepsin S mRNA levels were evident at a TNF- dose of 1.0
ng/ml. Maximal up-regulation of the MMP and cathepsin S mRNAs occurred
at doses of 10–20 ng/ml. In contrast, the higher concentrations of
TNF- did not affect TIMP-1 mRNA and reduced expression of the
primary 3.5-kb TIMP-2 transcript.
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treatment at a dose of 10 ng/ml from
three independent experiments are presented in Table 2
. The mean increases in MMP-1, -3, and
-9 and cathepsin S mRNAs were >10-fold.
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Stimulated Secretion of Matrix-Degrading Enzymes but not
TIMPs
Western blot analysis revealed increased levels of proMMP-1 (57
kd) and proMMP-3 (59 kd) in conditioned medium of CSMC treated with
TNF- (Figure 5)
. In contrast, levels
of proMMP-2 in the conditioned medium were unaffected by cytokine
treatment. The levels of TIMP-1 and -2 in the conditioned medium were
unaffected or lower, respectively, following cytokine treatment.
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(Figure 6)
. Gelatin zymography showed the
up-regulation of proMMP-9 (92- and 170-kd monomer and dimer). The
gelatin zymograms also confirmed that MMP-2 was unaffected by the
cytokine treatment.
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(10 ng/ml) for 48 hours. Active
cathepsin S (28 kd) was detected in cell lysates from control and
TNF--treated cells, whereas procathepsin S (~37 kd) protein was
detected only in conditioned medium from cultures treated with TNF-
(Figure 7)
.
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To determine whether the responses of CSMC to TNF- are
representative of responses to other pro-inflammatory cytokines, we
treated CSMC cultures with IL-1ß and examined expression of
MMP, TIMP, and cathepsin S mRNAs. IL-1ß treatment provoked a pattern
of gene expression that paralleled that observed following TNF-
treatment: MMP-1, -3, and -9 and cathepsin S mRNAs were increased,
whereas TIMP-1 and -2 mRNAs were not (Figure 8)
. Table 3
presents the change in MMP, TIMP, and cathepsin S mRNAs relative to
controls from three independent experiments in which CMSC were treated
with IL-1ß for 48 hours at a concentration of 10 ng/ml.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß, cytokines that may
participate in the normal process of cervical ripening or the premature
remodeling of the cervix associated with preterm labor.4,5
We also obtained preliminary evidence for CSMC expression of MMP-13, an
enzyme that degrades fibrillar collagens and also activates MMP-9
(unpublished observations). The abundance of MMP-13 mRNA was much lower
than that of MMP-1, -3, and -9 mRNAs but, like these other MMP
transcripts, MMP-13 message levels in the CSMC were increased by
cytokine treatment. These findings support the concept that resident
cells in the cervix, in addition to leukocytes that infiltrate the
cervix during cervical ripening, play roles in extracellular matrix
catabolism. The increase in MMP expression in response to pro-inflammatory cytokine treatment appears to be restricted to MMPs whose promoters contain AP-1 and Ets binding sites. These include the MMP-1, -3, and -9 and cathepsin S genes.16,30,31 In contrast, the MMP-2 gene does not contain these cis elements and MMP-2 expression was not increased by cytokine treatment. Thus, the cytokine-triggered up-regulation of expression of matrix-degrading enzymes may be the result of transcriptional activation mediated by a shared network of transcription factors.
The CSMC responded to TNF- with increased production of the
proenzyme forms of MMP-1, -3, and -9 and cathepsin S. These
proenzymes must be activated to catalyze matrix degradation and it is
presumed that activation of one or several of these proteinases (eg,
MMP-3 and MMP-13) may result in the activation of the others, as a
number of the MMPs have been reported to activate other members of the
MMP family.32
Our study does not disclose potential
mechanisms of proenzyme activation.
The activities of MMPs are restrained by TIMPs.33,34
It is
notable that although cytokine treatment increased expression of
certain MMPs, TIMP-1 and TIMP-2 expression was either unaffected or
reduced, dramatically shifting the ratio of enzymes to inhibitors.
Because we did not assess all four known members of the TIMP family of
proteins, or the expression of other enzyme inhibitors, including
2-macroglobulin and the cathepsin inhibitors, the cystatins, we do
not know whether the failure of TIMP-1 and TIMP-2 to respond to
cytokine treatment reflects a generalized shift in the balance of
proteinases to their respective endogenous inhibitors. It is notable
that Rechberger and Woessner35
reported dramatically
increased levels of collagenase activity in the cervix during labor
with a much less striking increase in TIMP-1 and 2-macroglobulin
levels. These observations are consistent with our findings on CSMC in
culture. Moreover, we have demonstrated cathepsin S and MMP-1 and -3
protein in CSMC in human cervical tissue removed at term by
immunohistochemistry (M. Watari, E.E. Furth, and J.F. Strauss, III,
unpublished observations). The latter findings demonstrate that these
enzymes are produced by CSMC in situ. The demonstration of
in situ expression of these enzymes is important because
smooth muscle cell migration in culture36
or serial cell
passage37
could influence MMP expression.
Our findings that pro-inflammatory cytokines increase expression of MMPs in CSMC are similar to, yet different from, reported studies on cervical fibroblasts.11,38 Pro-inflammatory cytokines increase MMP-1 and -3 mRNAs but not MMP-2 expression in both cell types. In contrast, TIMP expression is also increased in cervical fibroblasts but not in CSMC. This disparity may reflect fundamental differences between fibroblasts and smooth muscle cells.
In summary, we have obtained evidence supporting a key role for CSMC in restructuring of the cervical extracellular matrix and documented that pro-inflammatory cytokines can trigger the expression of a number of genes that can act in concert to hydrolyze fibrillar collagen and elastin. We propose that CSMC participate in the normal processes of cervical ripening as well as the premature cervical changes associated with preterm labor.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grant HD34612 (J.F.S.) and American Heart Association Scientist Development Grant 9730157N (G-P.S.).
Accepted for publication March 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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increases elastase-like enzyme in human uterine cervical fibroblasts. Gynecol Obstet Invest 1990, 30:239-241[Medline]
-induced gene expression of matrix metalloproteinases, and tissue inhibitor-1 of metalloproteinases (TIMP-1) in human uterine cervical fibroblasts. Biochim Biophys Acata 1996, 1220:57-65
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