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Department of Obstetrics, Gynecology, and Women’s Health, New Jersey Medical School, Newark, New Jersey 07103; and Isis Pharmaceuticals (B.P.M.), Carlsbad, California 92008
Address all correspondence and requests for reprints to: Dr. Laura T. Goldsmith, Department of Obstetrics, Gynecology, and Women’s Health, New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103. E-mail: goldsmit{at}umdnj.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The maintenance of connective tissue architecture requires a precise balance between the action of matrix metalloproteinases (MMPs), which degrade the extracellular matrix, and the endogenous tissue inhibitors of metalloproteinases, which regulate the activity of the metalloproteinases (18). The present studies were designed to determine the effects of relaxin on the regulation of human cervical MMP expression and to elucidate the mechanism of action of relaxin in these effects using an established in vitro model of human pregnancy cervix, lower uterine segment fibroblasts (19, 20, 21, 22, 23). As type I collagen is the major collagen type in human cervix, we evaluated the effects of relaxin on the expression of interstitial collagenase, stromelysin, and their endogenous inhibitor tissue inhibitor of metalloproteinase-1 (TIMP-1).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Regulation of MMP expression
The isolation, primary culture, and characterization of the
human lower uterine segment fibroblasts have been described in precise
detail previously (23). Briefly, lower uterine segment
tissue was removed at term pregnancy from a nonlaboring patient at the
time of cesarean section delivery following informed consent. The
protocol was approved by the institutional review board. Isolated,
cultured cells were characterized as fibroblasts free of contaminating
epithelial cells by immunohistochemistry of specific markers
(23). Cells were plated at 1 x
106 cells/well in 35-mm tissue culture dishes
(Becton Dickinson and Co., Lincoln Park, NJ) and
maintained in DMEM supplemented with 10% FBS, 5000 U/ml penicillin,
and 5000 µg/ml streptomycin sulfate (complete medium) overnight in an
atmosphere of 5% CO2/95% air. Medium was
removed from the cells and replaced with complete medium containing 1
µM 17ß-estradiol. This was repeated 24 and 48 h
later for a total of 72 h of estradiol priming. Medium was then
removed and replaced with serum-free DMEM supplemented with 0.2%
lactalbumin hydrolysate (LAH) without or with various amounts of
relaxin. Cells in replicate wells were also incubated with PMA (10
nM) as a positive control. After 65 h of incubation,
cells were removed from the plate using trypsin/EDTA and counted using
a Coulter counter (Miami, FL). Conditioned medium was maintained frozen
at -20 C until Western blotting was performed. Cells from replicate
wells were used to prepare RNA for Northern blotting.
Role of PKC in relaxin signaling
Cells were plated as described above. After 72 h of
estradiol priming, medium was removed and replaced with serum-free DMEM
supplemented with 0.2% LAH either without or with 0.5, 1, or 10 ng/ml
relaxin or 10 nM PMA. With each of these relaxin
concentrations as well as with the PMA group, two additional sets of
wells were treated with 0.2 µM CGP41251 or 0.2
µM CGP42700, respectively. The activities of these
compounds has been described previously (28). Cells were
incubated in an atmosphere of 5% CO2/95% air at
37 C for 65 h; the medium was then collected and frozen at -20 C
until Western blotting was performed. Cells were removed from the plate
by incubation with trypsin/EDTA and counted.
Role of receptor tyrosine kinase inhibition in relaxin
signaling
Cells were seeded at 1 x 105
cells/well in 24-well trays in complete DMEM and incubated for 48
h, after which they were serum-starved for 24 h. Subsequently,
they were pretreated with receptor tyrosine kinase inhibitors (5
µM HNMPA, 100 µM AG18, or 100
nM AG1478) in serum-free medium overnight. The next day,
cells were treated without or with either 1 nM relaxin, 10
ng/ml PDGF, or 10 ng/ml EGF (stimulators) in the absence or presence of
5 µM HNMPA, 100 µM AG18, or 100
nM AG1478 (inhibitors) as follows. Medium was replaced with
serum-free vanadate containing DMEM, and cells were incubated in the
absence or presence of stimulators with or without inhibitors for the
specified time periods, after which protein extracts were prepared and
subjected to phosphotyrosine Western blotting analysis as described
previously (23). For each stimulator, phosphorylation of
their respective receptor protein was assessed. HNMPA is a potent
inhibitor highly specific for tyrosine kinases over serine kinases
(29, 30).
Role of c-Raf in relaxin signaling
Two oligonucleotides that contain
2'-O-methoxyethyl/phosphodiester residues flanking a
2'-deoxynucleotide/phosphorothioate region that supports ribonuclease H
activation in cells, were used in these experiments, as used previously
(31, 32). The sequences are TCC-CGC-CTG-TGA-CAT-GCA-TT
(ISIS 12854) and TCC-CGC-CTA-CTA-CAT-GCA-TT (ISIS 14729, control for
ISIS 12854). ISIS 12854 has been used previously to inhibit c-Raf
protein expression in human cells (32). Cells were seeded
at 1.5 x 105 cells/well in 24-well trays.
The next day, cells were primed with 1 µM
17ß-estradiol for 48 h in complete medium. After 48 h, the
first oligonucleotide treatment was performed as follows. Cells were
washed with 0.5 ml EBSS, and 225 µl Opti-MEM containing 10 µl
Lipofectin/ml were added. Untreated, control cells received an
additional 25 µl Opti-MEM. Treated cells received 25 µl 2
µM oligonucleotide (final concentration, 200
nM) in Opti-MEM, either c-Raf antisense ISIS
12854 or c-Raf antisense mismatched ISIS 14729. Cells were then
incubated for 5 h at 37 C, after which the medium was aspirated
and replaced with complete medium containing 1
µM 17ß-estradiol, and cells were incubated
overnight. The next day, this complete protocol was repeated exactly as
described above. After this 5-h incubation, cells were washed once with
EBSS and fed with serum-free DMEM containing 0.2% LAH. Cells were then
incubated for 65 h at 37 C, without or with 10 ng/ml relaxin, and
conditioned medium was collected for MMP analysis by Western blotting.
For each category of cell treatment, the cells in one well were taken
for counting, and the remaining wells (three for each category) were
used in preparation of protein extracts.
Preparation of cell protein extracts for Western blotting
Cell protein extracts for Western blotting were prepared as
follows. Medium was aspirated, and the cells were rapidly washed with
EBSS. Cells were lysed by the addition of 50 µl nonreducing,
dye-free, boiling 2 x SDS-PAGE buffer/well. Lysates were
collected into small microfuge tubes (aided by scraping the wells with
a cell scraper) and boiled for 2 min. After cooling on ice, cell
lysates were passed through a 25-gauge needle four times to shear DNA.
An aliquot was removed for protein assay, and 5 µl 2-mercaptoethanol
and 0.5 µl 0.2% bromophenol blue solution (per 100 µl) were then
added to the extracts, which were boiled for 2 min and immediately
frozen at -20 C. Protein extracts were assessed for protein content
using the Protein Assay DC kit (Bio-Rad Laboratories, Inc., Hercules, CA) and BSA as standard.
Western blot analysis
SDS-PAGE and Western transfers were performed as previously
described (23, 33). Equivalent amounts of cell-conditioned
medium (10 µl medium heat treated with 5 µl 3 x SDS-PAGE
buffer, except for TIMP-1, for which 40 µl medium heat treated with
10 µl 5 x SDS-PAGE buffer were used) were used. For
phosphotyrosine immunoblotting, 10 µg protein equivalents of frozen
cell extracts (prepared as described) were used. Samples were loaded
onto 4% stacking and 10% (for MMPs), 12.5% (for TIMP-1 and c-Raf),
or 8% (for phosphotyrosine) separating gels and electrophoresed at 200
V for 45 min. Pure MMP and TIMP-1 proteins were used as controls, and
broad range, prestained kaleidoscope mol wt standards (Bio-Rad Laboratories, Inc.) were used to estimate the mol wt. Proteins
were electrotransferred at 100 V for 1.5 h onto PVDF membranes and
immunoblotted with specific antibodies. Membranes were blocked for 30
min with 3% BSA/Tris-buffered saline/0.1% Tween 20, pH 7.4, and
incubated with the primary antibodies overnight. The specificity of
these antibodies and their use in Western blot analysis to identify
human pro-MMP-1, pro-MMP-3, and TIMP-1 have been previously described
(34, 35, 36). After washing, membranes were incubated for
1 h with horseradish peroxidase-conjugated secondary antibodies
diluted in 3% BSA in Tris-buffered saline/0.1% Tween 20 (pH
7.4). Blots were developed by the enhanced chemiluminescence method as
described previously (23, 33) and exposed to
Kodak BioMax ML or MR film (Sigma).
RNA isolation and Northern blot analysis
Total RNA was isolated using the acid guanidinium
thiocyanate-phenol-chloroform extraction procedure (37).
Enrichment for poly(A)+ RNA was performed using
batch affinity chromatography on oligo(deoxythymidine) cellulose
(38). RNAs were denatured in the presence of formaldehyde
and formamide and fractionated on 1.2% agarose-formaldehyde gels. RNAs
were then transferred to nylon membranes (Roche,
Indianapolis, IN) using a Turbo-Blotter rapid downward transfer
apparatus (Schleicher & Schuell, Inc., Keene, NH) in
20 x SSC. RNAs were fixed by UV cross-linking, and the membranes
were probed with 32P-labeled cDNAs or
oligonucleotides. Blots were prehybridized in buffer containing 50%
formamide, 10% dextran sulfate, 0.5% SDS, 1 M NaCl, and
5 x Denhardt’s solution at 42 C for 2 h in a glass bottle
in a minihybridization oven (Hybaid, Labnet, Woodbridge, NJ). Denatured
labeled probe was added to the buffer, and the membranes were incubated
for an additional 18 h at 42 C. Membranes were then washed twice
in 2 x SSC at 42 C for 5 min each time, followed by two washings
in 2 x SSC-1% SDS at 60 C for 30 min each time, followed by two
washings in 0.1 x SSC at room temperature for 30 min each time.
After washing, blots were subjected to autoradiography. cDNAs were
labeled by random priming with
[
-32P]deoxy-CTP using the Random Primers DNA
Labeling System (Life Technologies, Inc., Grand Island,
NY) following the instructions supplied by the manufacturer. The TIMP-1
oligomer was end labeled with [
-32P]ATP
using T4 polynucleotide kinase (New England Biolabs, Inc.,
Beverly, MA). Labeled probes were separated from free nucleotides using
Chroma Spin+ TE-10 Columns (CLONTECH Laboratories, Inc., Palo Alto, CA). Blots were exposed to
Hyperfilm-MP (Amersham Pharmacia Biotech, Arlington
Heights, IL) at -80 C.
Densitometric analysis
The intensities of the signals obtained on developed films were
determined using a computing densitometer (300B, Molecular Dynamics, Inc., Sunnyvale, CA) using the volume integration
method with appropriate corrections for background absorption, as
described previously (23, 33).
Statistical analyses
Densitometric values for control untreated cells were
arbitrarily set at 100, and values for treatment groups are expressed
as a percentage of the control. All data generated (except for the PKC
inhibitor studies) were normally distributed, as assessed using the
Shapiro-Wilk test, and of equal variances, and thus were compared
parametrically using two-tailed t tests. Control and
treatment values from experiments using the PKC inhibitor and the PKC
inhibitor analog were not normally distributed; therefore, analyses of
these data were performed nonparametrically using Kruskal-Wallis
rank-sum testing. All comparisons were performed using JMP statistical
software (SAS Institute, Inc., Cary, NC) written for
the Macintosh Computer (Apple Computers, Cupertino, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Both 1 and 10 ng/ml relaxin
significantly increased procollagenase protein expression to mean
(±SEM) levels of 204 ± 26% (P =
0.006) and 211 ± 21% (P = 0.02; n = 5
experiments for each dose) of the control, respectively. Relaxin (10
ng/ml) also caused a significant increase in the expression of the
2.2-kb procollagenase mRNA transcript to 214 ± 35% (n = 4
experiments; P = 0.04) above the control value, as
shown in Fig. 1B.
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. Both 1 and 10 ng/ml relaxin
significantly increased prostromelysin expression to mean levels of
241 ± 46% (P = 0.04) and 198 ± 31%
(P = 0.04; n = 5 experiments for both) of the
control, respectively. Relaxin at 10 ng/ml also caused a significant
increase in the expression of the 1.9-kb prostromelysin mRNA transcript
to 189 ± 26% (n = 4 experiments; P = 0.04)
above the control value, as shown in Fig. 2B.
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, relaxin at
0.5, 1, or 10 ng/ml significantly decreased TIMP-1 expression to
67 ± 9% (P = 0.02), 50 ± 7%
(P = 0.001), and 45 ± 5% (P <
0.001) of the control value, respectively (n = 6 experiments). In
contrast to its effect at the level of the protein, relaxin at 10 ng/ml
did not affect steady state levels of the 0.9-kb TIMP-1 mRNA transcript
(data not shown).
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, treatment of human lower uterine segment fibroblasts with 0.1, 1, 10,
100, or 1000 nM progesterone significantly decreased
procollagenase protein expression to 63 ± 4%, 58 ± 4%,
54 ± 10%, 52 ± 5%, and 57 ± 3% (mean ±
SEM; n = 4 experiments; P < 0.05 at
all amounts) of the control value, respectively. Expression of
prostromelysin protein levels was similarly inhibited by these amounts
of progesterone to 59 ± 13%, 53 ± 9%, 58 ± 8%,
60 ± 4%, and 64 ± 8% (mean ±
SEM; n = 4 experiments; P <
0.05 at all amounts) of the control, respectively. In contrast,
expression of TIMP-1 protein levels was not affected by treatment with
progesterone at these doses. Levels remained at 100 ± 4%,
93 ± 9%, 96 ± 11%, 106 ± 9%, and 117 ± 11%
(mean ± SEM; n = 4 experiments;
P > 0.05 at all amounts) of the control,
respectively.
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, treatment of
the cells with 1 or 10 ng/ml relaxin caused a significant increase in
MMP-2 expression to 210 ± 31% (mean ± SEM;
n = 4 experiments; P = 0.039) and 193 ± 18%
(P = 0.014) of the control value, respectively. The
increase in MMP-2 expression caused by treatment of the cells with 0.5
ng/ml relaxin was less pronounced (154 ± 25%; P
= 0.11). In contrast, MMP-2 protein levels were not affected by
incubation with progesterone at 0.1, 1, 10, 100, or 1000
nM. Levels remained at 109 ± 20%, 117
± 15%, 107 ± 13%, 94 ± 7%, and 97 ± 10%
(mean ± SEM; n = 4 experiments;
P > 0.05 at all amounts) of the control,
respectively.
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shows a representative phosphotyrosine Western blot of total cellular
proteins from cells incubated in the absence or presence of 1
nM relaxin without or with 5 µM HNMPA. Figure 6B shows the reproducibility and consistency of this finding. HNMPA
significantly inhibited relaxin-stimulated tyrosine phosphorylation of
the approximately 220-kDa protein to 33.3 ± 8%
(±SEM; P = 0.004; n = 4 experiments)
and 39.2 ± 7% (P = 0.003; n = 4
experiments) of uninhibited, relaxin-stimulated levels at 15 and 30 min
of treatment, respectively, similar to nonrelaxin treated levels at the
same treatment times. In contrast, two other receptor tyrosine kinase
inhibitors, selective for PDGF (AG18) and EGF (AG1478), had no effect
on relaxin-stimulated 220-kDa protein tyrosine phosphorylation, as
shown in Table 1. Demonstration of the
inhibitory activity of AG 18 and AG 1478 on PDGF and EGF receptor
phosphorylation (positive controls) is also shown in Table 1.
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) and 61.2 ± 1.5% (P =
0.0016) of mismatched oligonucleotide-treated levels in untreated and
relaxin-treated cells, respectively (Fig. 7A). Inhibition of c-Raf
protein resulted in significant down-regulation of relaxin-stimulated
pro-MMP-1 to 60.5 ± 3.3% (P = 0.007; Fig. 7B)
and of relaxin stimulated pro-MMP-3 protein levels to 56 ± 8.8%
(P = 0.037; Fig. 7C) of those in relaxin-treated cells
transfected with the mismatched oligonucleotide control. Reduction of
c-Raf protein levels also resulted in a reversal of relaxin inhibition
of TIMP-1 protein levels to 146 ± 3.3% (P = 0.005) of
those in relaxin-treated cells transfected with the mismatched
oligonucleotide control (Fig. 7D). Expression of pro-MMP-1, pro-MMP-3,
and TIMP-1 protein levels in nonrelaxin-treated cells were not altered
by inhibition of c-Raf protein levels (Fig. 7, B–D).
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, the highly selective PKC inhibitor
used in these experiments markedly inhibited PMA-stimulated pro-MMP-1
and pro-MMP-3 expression, yet no effect on relaxin-stimulated
levels was observed. A structurally similar, yet inactive, compound was
used as a negative control (28).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Relaxin action on certain target organs, for example, the rat uterus, is synergistic with the actions of progesterone (39). However, other data suggest that this may not be the case in all target organs. To determine whether relaxin and progesterone have similar or different actions on metalloproteinase expression in the human cervix, we determined the effects of progesterone on the expression of MMPs and TIMP-1. Our data demonstrate that progesterone action on MMP expression in human lower uterine segment fibroblasts is opposite that of relaxin. Progesterone negatively regulates MMP expression. In the present studies progesterone inhibited the expression of pro-MMP-1 and pro-MMP-3 and had no effect on TIMP-1.
As a hallmark of cervical dilatation at term pregnancy in women involves infiltration of leukocytes, we examined the effect of relaxin on MMP-2 (gelatinase A), which degrades basement membrane collagens, elastin, laminin, and fibronectin (18). The data presented here demonstrate that relaxin significantly stimulates the expression of MMP-2, suggesting a mechanism by which relaxin may foster the infiltration of leukocytes and other cell types into the cervix. This effect of relaxin could serve to enhance the direct positive regulatory effect of relaxin on MMP expression by increasing the local concentrations of various MMP stimulatory cytokines secreted by these cells. The stimulatory action of relaxin on MMP-2 expression in this cell type differs from that of progesterone, which the present data demonstrate has no effect.
We previously demonstrated that the response of human lower uterine segment fibroblasts to relaxin is associated with an increase in tyrosine phosphorylation of an approximately 220-kDa protein, probably the relaxin receptor (23). To advance our understanding of the mechanisms of relaxin signaling in the cervix and test the hypothesis that the relaxin receptor is a tyrosine kinase receptor, the effects of inhibition of receptor tyrosine kinase activity on relaxin-stimulated tyrosine phosphorylation of the 220-kDa protein were studied. Treatment with a potent, specific receptor tyrosine kinase inhibitor (29, 30) demonstrated a significant inhibition of relaxin-stimulated tyrosine phosphorylation of the 220-kDa protein. In contrast, two other receptor tyrosine kinase inhibitors, selective for PDGF (AG18) and EGF (AG1478), had no effect on relaxin-stimulated tyrosine phosphorylation. These data support the concept that the relaxin receptor is a tyrosine kinase receptor distinct from the PDGF and EGF receptors.
The most well characterized signaling cascade used by tyrosine kinase receptors bound to ligand involves the successive activation of Ras, c-Raf, mitogen-activated protein kinase kinase 1 and 2 (MEK) and mitogen-activated protein kinases (ERK1 and ERK2). To elucidate the components of the pathway that is used in relaxin signaling, we studied the effect of specific inhibition of synthesis of c-Raf kinase. Transfection with a specific c-Raf mRNA-targeted antisense oligonucleotide (31, 32) resulted in effective inhibition of c-Raf protein levels, which, in turn, caused a significant down-regulation of relaxin-stimulated pro-MMP-1 and relaxin-stimulated pro-MMP-3 protein levels. Reduction of c-Raf protein levels also resulted in a reversal of relaxin inhibition of TIMP-1 protein levels. Expression of pro-MMP-1, pro-MMP-3, and TIMP-1 protein levels in nonrelaxin-treated cells was not altered by inhibition of c-Raf protein levels. These data point to the involvement of c-Raf in relaxin signaling and, as MEK is the only known kinase directly downstream of c-Raf, suggest the utilization of a MEK-ERK kinase cascade. However, some evidence supports the concept that Raf can also activate other effectors (40, 41, 42). The complete pathway used by relaxin requires further study.
Thus, the present data demonstrate that relaxin appears to positively regulate MMP expression in human lower uterine segment fibroblasts using a signaling pathway that involves a tyrosine kinase receptor and c-Raf kinase. Our previous data, which demonstrate that relaxin does not increase intracellular levels of cAMP in human lower uterine segment fibroblasts, suggest that the relaxin receptor is not a heterotrimeric G protein-associated receptor (23). Although increased intracellular cAMP concentrations in several cell types are associated with relaxin treatment (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17), to date no data have been provided by any studies that demonstrate coupling of a relaxin-receptor complex to a heterotrimeric G protein-coupled receptor.
Various cytokines and other stimulators of MMP expression in several cell types use PKCs in mediating their effects (43). In contrast, inhibition of the activity of PMA-sensitive PKCs did not influence relaxin stimulation of pro-MMP-1 and pro-MMP-3 protein levels. Inhibition of PKC activity in these experiments markedly inhibited PMA-stimulated pro-MMP-1 and pro-MMP-3 expression, yet had no effect on relaxin-stimulated levels. The structurally similar inactive compound demonstrated no effect on relaxin-regulated expression of pro-MMP-1 or pro-MMP-3 or upon PMA-stimulated pro-MMP-1 and pro-MMP-3 expression. These data demonstrate that relaxin stimulation of MMPs does not involve PMA-sensitive PKCs. However, the involvement of recently described PMA-insensitive PKCs (44) remains unknown. Involvement of a PMA-insensitive isotype has been shown in PDGF activation of the stromelysin promoter (45).
Our data and those of others suggest that relaxin action on connective
tissue may be unique. Cytokines such as interleukin-1 stimulate the
production of MMP-1 and MMP-3 in human cervical fibroblasts in
vitro (35). However, unlike relaxin, which inhibits
the expression of TIMP-1 in our model system and in dermal fibroblasts
(46), interleukin-1 increases TIMP-1 production in
human cervical fibroblasts and in fibroblasts from other organs
(34, 46, 47, 48). Other growth factors and cytokines, such as
EGF and TNF also stimulate the production of MMP-1, and MMP-3.
However, in addition, these agents either stimulate TIMP-1 production
in human fibroblasts or have no effect (36, 49). Other
factors, such as retinoids, interferons, and glucocorticoids, inhibit
TIMP-1 expression, yet they inhibit MMP expression as well
(50). Thus, relaxin may be unique in its ability to have
inhibitory effects on the endogenous inhibitor of MMPs while
stimulating the MMPs. This activity of inhibition of TIMP-1 expression
by relaxin may account for the more modest stimulatory effects of
relaxin on MMP-1 and MMP-3 expression compared with the effects of
cytokines such as interleukin-1, which also stimulate levels of the
inhibitor. This may have a biological basis; a more modest effect of
relaxin may be necessary to allow connective tissue remodeling, yet not
cause the kind of connective tissue destruction that may occur in
reactions mediated by inflammatory cytokines. Unlike relaxin, MMP
stimulatory activities of cytokines such as interleukin-1 require
PMA-sensitive PKCs in certain cell types, including human cervical
fibroblasts (34). Yet, similar to relaxin, cAMP is not a
second messenger for interleukin-1 action in this system
(34). Certain aspects of relaxin signaling that regulate
MMP expression in human lower uterine segment fibroblasts may be common
to those of other regulators. For example, stromelysin-1 gene induction
by PDGF is dependent upon Ras, which then directs two distinct
pathways, one of which is c-Raf dependent (which may resemble relaxin
signaling) and another of which is c-Raf independent. Cytokines and
growth factors have been shown to regulate the production of each MMP
and endogenous tissue inhibitor using many different pathways in a
ligand-, developmental, and tissue-specific manner. Thus, understanding
of the complete signaling pathway and even multiple paths that
regulate relaxin signaling will require additional study in this cell
type.
The effects of relaxin on cervical connective tissue must be considered in relation to the actions of the steroid hormones, estrogen and progesterone. In contrast to the previously demonstrated synergistic effect of relaxin and progesterone on the uterus (39), relaxin and progesterone appear to have opposing influences on cervical MMP expression, which may maintain a balance that results in a net effect of maintaining cervical connective tissue integrity during pregnancy. At term pregnancy in women or during premature labor, alterations in progesterone metabolism or progesterone action, such as that proposed to be caused by repression of the progesterone receptor, may allow the balance to be shifted to a more pronounced effect of relaxin in concert with a lesser effect of progesterone, resulting in the rearrangement of connective tissue caused by the resultant relative increase in MMP activity and decrease in TIMP-1 activity. The present data allow for the hypothesis that progesterone may to some extent block or prevent the effects of relaxin on the cervix. A greater effect of relaxin may thus occur when progesterone concentrations are decreased or when the action of progesterone is suppressed. Thus, in women, relaxin action on the cervix may be more robust at term pregnancy when progesterone biological activity may be decreased. Also, at very high concentrations, relaxin may overcome this blockage by progesterone, causing an increase in prematurity risk.
The mechanisms used to accomplish cervical ripening during human pregnancy are extremely complex and require the actions of multiple interacting peptide and steroid hormones and other factors and cellular responses. The evidence that relaxin is a positive regulator of the MMPs that affect type I collagen, in contrast to the negative effects of progesterone, and that relaxin stimulates pro-MMP-2 provides a potential explanation for the relationship between increased circulating maternal levels of relaxin and the increased risk of prematurity. In addition, these findings allow for the formulation of a new hypothesis regarding the role of the relative actions of relaxin and progesterone at term delivery. The collective data now suggest that the relaxin receptor is a tyrosine kinase receptor that, when bound to relaxin, activates a signaling pathway(s) mediated by the c-Raf kinase, a pathway that does not involve PMA-sensitive PKCs or cAMP.
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Acknowledgments |
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Footnotes |
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1 Present address: Department of Obstetrics and Gynecology,
Roosevelt-St. Lukes Medical Center, 1000 Tenth Avenue, New York, New
York 10019. 
2 Present address: Reproductive Endocrine Unit, Massachusetts
General Hospital, 55 Fruit Street, Boston, Massachusetts
02114.
Abbreviations: EBSS, Earle’s Balanced salt solution; EGF, epidermal growth factor; HNMPA, hydroxy-2-naphthalenyl methyl phosphonic acid trisacetoxymethyl ester; LAH, lactalbumin hydrolysate; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; PMA, phorbol-12-myristate-13-acetate; TIMP-1, tissue inhibitor of metalloproteinase-1.
Received November 27, 2000.
Accepted for publication April 4, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induces mRNA for collagenase and TIMP in human skin fibroblasts.
Connect Tissue Res 25:161–170[Medline]
-induced gene expression of
matrix metalloproteinases and tissue inhibitor-1 of metalloproteinases
(TIMP-1) in human uterine cervical fibroblasts. Biochim Biophys Acta 1220:57–65[Medline]
PKC
plays a critical role during stromelysin promoter activation by
platelet-derived growth factor through a novel palindromic element.
J Biol Chem 269:10044–10049
induces mRNA for collagenase and TIMP in human skin fibroblasts.
Connect Tissue Res 25:161–170
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