Vol. 81, No. 9
Printed LIZ U.S.A.
0021-972x/96/$03.00/0
Journal
of Clinical
Endocrmology
and Metabolism
Copyright
0 1996 by The Endocrine
Society
Relaxin
Activity*
Modulates
JIUAN-JIUAN
HWANG,
Department
of Environmental
University School of Medicine,
DAVE
Human
MACINGA,
AND
Cervical
ELLEN
Health Science and Reproductive
Cleveland, Ohio 44106
Stromal
Cell
A. RORKE
Biology,
Case Western Reserve
lated the secretion
of several
cervical
stromal
proteinase
activities,
including
36, 52, and 116 kDa. Inhibitor
studies using ethylenediamine tetraacetate,
l,lO-phenanthroline,
and L-cysteine
identify
these
gelatinases
as metalloproteinases.
Neither
estradiol
nor progesterone
augmented
the effects of relaxin.
A relaxin-induced
concentrationdependent
increase
in collagenase
activity
was observed
when measured with a conventional
collagen
fibril assay. Finally,
relaxin
was
found to increase glycosaminoglycan
synthesis,
as indicated
by [3H]glycocyamine
incorporation
in human
cervical
stromal
cell cultures.
These results indicate
that relaxin
may regulate
cervical
ripening
in
humans,
as it does in other species, by direct effects on cervical cells.
(J Clin Endocrinol Metab 81: 3379-3384,
1996)
ABSTRACT
The cervix
undergoes
profound
changes
in size and consistency
during pregnancy
which are designed
to facilitate
a normal
delivery.
These changes are under the control of a number
of hormonal
factors.
Experimental
and clinical
studies
suggest
that relaxin,
a protein
hormone,
plays a role in promoting
cervical
softening.
One possible
target site for the effects of relaxin
on the cervix is the stroma.
In the
studies reported
here, cultures
of normal human cervical stromal
cells
were used to determine
what direct effects relaxin
has on cervical
cells. Relaxin
at concentrations
ranging
from 0.10-100
ng/mL had no
effect on human
cervical
stromal
cell proliferation,
but was found to
modulate
stromal
cell activity
related
to the remodeling
of extracellular matrix.
When gelatin
substrate
gels were used, relaxin
stimu-
I
N HUMANS,
cervical
ripening
takes place during the last
few weeks of pregnancy
(1, 2), and disorders
in this
process cause obstetric complications,
potentially
endangering both fetus and mother. Cervical maturation
is associated
with changes in proteinase
activity (3-6) and the composition of the extracellular
matrix (Y-14).
Relaxin, a 6000-dalton
dimeric polypeptide
hormone, has
been demonstrated
to stimulate cervical softening in a number of species, including
rodents (15-24) and domestic animals (16,21,25-30).
Animal studies show that porcine relaxin
treatment induces cervical extensibility
(15-18, 25, 26), decreases delivery duration
time (29,31), increases the number
of live pups (29,31,32),
and reduces the retention of fetuses
in utero (29,31,32). Also, despite having normal pregnancies,
mice with Hertwig’s
anemia (i.e. homozygous
for the mutant
allele an/ an) are nonresponsive
to relaxin and are unable to
deliver their pups (33). I n women, the corpus luteum is the
major source of relaxin; it has also been detected in decidua,
placenta, and endometrium
(34-38). During the first trimester, luteal secretion and peripheral
blood levels of relaxin
increase and remain elevated throughout
pregnancy (21,39).
Clinical studies using either porcine or recombinant
human
relaxin have showed limited effectiveness in stimulating
cervical ripening
(40-45). To date, four clinical trials in women
demonstrate
that porcine relaxin given either intravaginally
or intracervically
promotes
cervical ripening
and labor induction near term (40-43). Nevertheless,
a recent study using a single dose of recombinant
human relaxin showed no
significant
improvement
in cervical ripening
in women compared to that in the placebo group (45).
To elucidate how relaxin may influence cervical maturation, its effect on homogeneous
populations
of human cervical stromal cells was examined.
As cervical ripening
is
believed to result from collagenaseand proteinase-dependent breakdown
of the stromal matrix, changes in these
components
were examined
after relaxin treatment.
Human
cervical
Materials
and Methods
stromal cell culture
Human
cervical tissue was obtained
from women
undergoing
hysterectomy
for a variety
of uterine
disorders.
The procurement
procedures for the collection
of discarded
tissue was approved
by the human
studies review
board of University
Hospital.
The cervix was washed
with Hanks’ Balanced
Salt Solution
(HBSS), trimmed
of the epithelial
and smooth muscle components,
cut into 2-mm2 blocks, and washed
with HBSS. The tissue (8-10 pieces) was placed into dishes with culture
medium.
Within
a week, spindle-shaped
cells were observed
growing
outward
from each explant.
These cells were grown
to confluence,
at
which time they were harvested
with trypsin
and either frozen or subcultured.
Cells were grown in DMEM
supplemented
with penicillin
(100
LJ / mL), streptomycin
(100 mg / mL), L-glutamine
(2 mm01 / L), insulin (5
wg/mL),
transferrin
(10 kg/mL),
and 10% FCS. Near-confluent
cultures
of third to fifth passage cells were used in all experiments.
Cells were
identified
as stromal
based on the absence of immunnoreactivity
to
antikeratin
antibody.
Keratins
were expressed
only in epithelial
cells.
The cervical stromal cells were immunopositive
for vimentin.
In experiments involving
proteinase
activity,
the cells were shifted to a serumfree medium
containing
the same additives
plus 0.2% lactalbumin
hydrolysate.
Purified
porcine relaxin (NIH-R-PI)
was obtained
from the National
Received
September
25, 1995. Revision
received
March 4, 1996. Accepted April 5, 1996.
Address
all correspondence
and requests
for reprints
to: Ellen A.
Rorke,
Ph.D., Department
of Environmental
Health
Sciences,
Case
Western
Reserve
University
School of Medicine,
Room WG-19,
2109
Adelbert
Road, Cleveland,
Ohio 44106-4940.
*This work was supported
in part by the Ohio Cancer Research
Associates
and NIHES Grant 05227.
t Current
address:
Jiuan-Jiuan
Hwang,
Institute
of Physiology,
National
Yang-Min
University,
Taipei, Taiwan.
3379
HWANG
3380
Hormone
and Pituitary
Program.
Relaxin
diluted
to loo-fold
stock concentrations.
Cell proliferation
was reconstituted
in water
and
assays
Cells were seeded at l-2.5 x lo4 cells/mm’
and allowed
to attach
overnight.
At this time (day 0), triplicate
wells were counted.
The remaining
wells were washed once with HBSS, and medium
containing
1% FCS and the desired concentration
of relaxin was added to the wells.
Cells were allowed
to grow for up to 9 days, after which they were
washed
twice with HBSS, harvested
with trypsin,
and counted
in a
Coulter
counter
(Coulter
Electronics,
Hialeah, FL).
Visualization
of proteinase
gelatin substrate gels
activities
on polyacrylamide
Gelatin
zymography
was performed
as previously
described
with
slight modifications
(46,47). Gelatin was used as substrate because it is
readily cleaved by connective
tissue proteinases
and is easily incorporated into the polyacrylamide
gel. Samples of culture media were mixed
with Laemmli
sample buffer without
reducing
agent and electrophoresed without
boiling under nonreducing
conditions
on a Mini-Slab
gel
apparatus.
Medium
samples loaded onto the gel were normalized
based
on cell number.
Gels were run at 15 milliamperes/gel
in the stacking
phase and at 20 milliamperes/gel
during
the resolving
phase at 4 C.
After electrophoresis,
the gels were soaked in 2.5% Triton X-100 with
gentle shaking
for 30 min at room temperature
with one change of
detergent
solution.
This step removes
the SDS from the gel and allows
the enzyme
to recover activity.
Gels were rinsed and incubated
overnight at 37 C in substrate
buffer (50 Mm Tris-HCl,
pH 8, containing
5
mmol/L
CaCl, and 0.02% NaN,). After incubation,
the gels were stained
with 0.5% Coomassie
blue R-250 in acetic acid-isopropyl
alcohol-water
(1:3:6), destained
in water,
photographed,
and quantitated
by laser
densitometry.
Quantitation
of collagenase
activity
Collagenase
activity released into culture medium
was assayed using
the [i4C]collagen
fibril assay (48, 49). The collagen
substrate
was obtained by acetylation
of type I collagen using [r4C]acetic anhydride.
One
hundred
microliters
of acetylated
collagen (1 mg/mL
solution
in TrisHCl buffer, pH 7.6, containing
200 mmol/L
NaCl and 0.03% toluene)
were added to each microfuge
tube and incubated
at 35 C for 16-20 h
to allow fibril formation.
Samples of media made up to 100 PL with
water and 100 PL buffer (100 mmol/L
Tris-HCl,
pH 7.6, containing
15
mmol/L
CaCl,) were added to the collagen
fibrils and incubated
with
constant
shaking
in a 35 C water bath for 20 h. In every experiment
control
tubes containing
only buffer or 10 pg trypsin
were included.
Total lysis of the collagen was determined
for each experiment
by adding
10 pg Clostridial
collagenase
to control tubes. After digestion,
samples
were centrifuged
at 10,000 X g for 10 min to remove undigested
collagen
fibrils, and the supernatant
was counted in a liquid scintillation
counter.
Collagenase
activity in the samples was defined as (sample cpm - blank
cpm) / (total clostridial
collagenase
cpm - blank cpm).
Determination
of glycosaminoglycans
Incorporation
of [3H]glucosamine
into cells and extraction
of glycosaminoglycans
were performed
as described
by Fukui et al. (50). Five
microcuries
of o-[6-3H]glucosamine
hydrochloride
and 0.1-100 ng/mL
porcine relaxin or vehicle were added to the cell cultures (use of [3H]glucosamine allows both hyaluronic
acid and sulfated glycosaminoglycans
to be labeled), and the cells were incubated
for 24 h at 37 C. Both medium
and cell layer were subjected to papain digestion.
Papain proteolysis
was
carried
out for 48 h at 37 C. The resulting
digests were subjected
to
alkaline
hydrolysis
in 0.5 mol/L
NaOH
for 24 h at 4 C and then neutralized
with HCl. Proteins in the samples were removed
by trichloroacetic acid precipitation
and centrifugation.
Glycosaminoglycans
were
precipitated
from the supernatants
using 4 vol 95% ethanol
solution
containing
1% (wt/vol)
potassium
acetate, stored at 4 C for 24 h, and
centrifuged
at 10,000 rpm for 30 min at 4 C. The precipitates
were washed
with 70% ethanol
and dried in a speed vacuum
drier, and the dried
ET AL.
JCE & M . 1996
Vol81 . No 9
material
was dissolved
was used to determine
in a small volume of distilled
the total counts incorporated.
Statistical
analysis
data
analyzed
using
Data were
two-tailed
Student’s
water.
An aliquot
t test.
Results
Human cervical stromal cells exhibited fibroblastic morphology when cultured in vitro (Fig. lA), and these cells,
which doubled nearly every 48-72 h (Fig. lB), were not
induced to proliferate when exposed to relaxin (Fig. 1C). As
it is believed that cervical stromal fibroblasts play an important role in the reorganization of cervical connective tissue during pregnancy and labor, we evaluated the effects of
relaxin on cervical stromal proteinase activity, collagenase
activity, and glycosaminoglycan synthesis, becausetheseparameters are believed to contribute to cervical ripening.
Porcine relaxin increased proteinase activities secreted by
cultured human cervical stromal cells compared to control
values. As shown in Table 1, there are two major proteinase
activity bands, with approximate molecular massesof 116
and 36 kDa, that are significantly enhanced or induced by
relaxin treatment. This increase was dose dependent with an
optimal dose of 100 ng relaxin/mL (Table 1). The effect was
specific for relaxin, becauseneither progesterone (1 pmol/L)
nor estradiol (40 nmol/L) altered proteinase activities (Fig.
2). In addition, neither steroid augmented the effects of relaxin (data not shown). Two other bands (52 and 57 kDa)
were induced in cultures treated with 100 ng / mL relaxin. A
fifth band (180kDa) was unaffected by relaxin treatment. The
time course of induction was evaluated over a 6-day treatment period. A maximal stimulatory effect of relaxin (100
ng/mL) on the secretion of 52-kDa proteinase was observed
on day 2, the earliest time point examined (Fig. 3). The effect
of relaxin on the other proteinases displayed a similar time
course (data not shown). The activities of all proteinaseswere
inhibited by ethylenediamine tetraacetate, l,lO-phenanthroline, and L-cysteine, indicating that they are metalloproteinases(51).
The enzymatic breakdown of collagen, through the action
of collagenaseand other proteinases, is believed to constitute
in part the cellular basis of cervical softening before labor
(10). In Fig. 4, we demonstrate the collagenase activity of
control and relaxin-treated cells. Cervical stromal cells secreted a collagenase activity into the medium, and this activity was stimulated 2- to 3-fold when cells were treated
with relaxin.
Cervical maturation was associated with changes in the
organization of the extracellular matrix (23, 52). The major
constituents of the extracellular matrix are glycosaminoglycans that consist of repeating disaccharide units of a hexosamine and a hexuronic acid / hexose. Glycosaminoglycans
are often sulfated and negatively charged. Proteoglycans are
composed of glycosaminoglycan chains covalently attached
to a core protein. The nature of the proteoglycan is mainly
determined by its glycosaminoglycan moiety. These molecules are important determinants of the physical structure
and physiological function of the cervix. We used [3H]glucosamineincorporation into glycosaminoglycans to measure
RELAXIN
MODULATES
CERVICAL
STROMAL
CELL ACTIVITY
3381
TABLE 1. Proteinase Activity (percent control)
Mol mass
&Da)
36
52
57
116
180
0.1
108 t 7.3
102 ? 3.9
99 + 3.5
117 -c 5.9
100 2 6.1
Relaxin cont. (ng/mL)
1.0
10
120 t 7.1
140 2 9.7”
110 ir 2.7
118 t 7.1
97 t 3.0
100 -c 2.4
128 -c 7.6”
141 + 5.7”
98 + 7.4
105 t 2.5
200
152
115
198
107
100
t 6.3”
t 6.8”
-c 7.0”
t 8.8”
t 3.8
Zymogen analysis was performed as described in Materials
and
Bands were scanned with a laser densitometer, and the data
are presented as a percentage of the control value and the mean t SD
(n = 3 independent determinations).
a Statistically significant, P < 0.05.
Methods.
c E, p ho boo
B %
l- 50
x
ii
40
s
$
30
20
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10
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0
4a360
2
4
6
DAYS
8
10
FIG. 2. Effects ofrelaxin on proteinase activities secreted by cultured
human cervical stromal cells: a representative gelatin zymogram.
Cultures were treated with porcine relaxin (R; 10 or 100 ng/mL),
progesterone (P; 1 pmol/L), estradiol (E,; 40 nmoliL), or vehicle(C) for
7 days. Proteinase activity present in the culture medium was monitored by electrophoresis in denaturing polyacrylamide gels impregnated with gelatin as described in Materials and Methods.
Samples
were normalized based on cell number.
cosaminoglycans (Fig. 5). The identity of the individual glycosaminoglycans awaits further characterization.
Discussion
0
1
Relaxin
10
100
(nglml)
FIG. 1. A, Phase contrast microgram of cervical stromal cells showing
fibroblastic morphology. B, Proliferation of human cervical stromal
cells in culture. Cells were plated at 2.5 X lo4 cells/cm’ and allowed
to attach for 24 h (day 0). Cells from triplicate wells were harvested
on days 0, 3,5, 7, and 9 and counted on a Coulter counter. C, Effects
of relaxin on cervical stromal cell growth. Cultures were treated with
various doses of porcine relaxin for 7 days. The conditions of cell
culture and determination
of total cell number are described in
Materials and Methods. The results are presented as the mean k SEM
(n = 3).
in glycosaminoglycan
synthesis. Relaxin at l-100
ng/mL stimulated [3H]glycocyamine incorporation in a
dose-dependent manner in both cellular and secreted glychanges
Experimental and clinical studies indicate the importance
of relaxin, a polypeptide hormone, in promoting cervical
ripening during late pregnancy in animals and humans (19,
22, 30, 40-43). Evidence linking relaxin to cervical maturation during pregnancy in humans include elevated peripheral blood levels during late pregnancy (21, 39), increased
relaxin bioactivity levels in human cervical tissue extracts at
term (ll), the demonstration that highly purified porcine
relaxin given near term promotes cervical maturation (4043), and results indicating an association between placenta
relaxin deficiency and cervical dystocia (53). Relaxin, therefore, is a candidate to induce cervical maturation in women
who come to term with an unripened cervix (44, 45). Elucidation of the biochemical mechanismsand the cellular basis
by which relaxin promotes cervical maturation is important
if it is to be optimally applied for prevention of the above
clinical problems. We have developed a cervical stromal cell
culture system as a model to test relaxin’s direct effects on
human cervical stromal cell function.
The cultured human cervical cells retain a fibroblast phe-
HWANG
3382
g
250
:
5
F
Y
u
200
150
:
100
3
5
50
5
0
0
2
4
6
DAYS
FIG. 3. Time course of effects of relaxin
on proteinase
activity
secreted by cultured
human
cervical
stroma
cells. Cells were treated
with 100 ng/mL porcine relaxin
or vehicle for up to 6 days. Data are
presented
as the mean % SEM percentage
of control
activity
for the
52-kDa
band, which is representative
of all of the activities
increased
by relaxin.
TI
0
1
Relaxin
10 100
(rig/ml)
FIG. 4. Effects of relaxin
on collagenase
activity
secreted by cultured
human
cervical stroma cells. Cells were treated
with porcine relaxin
(l-100
ng/mL)
for 24 h. The collagenase
activity
present
in the conditioned
medium was analyzed
using a [r4Clcollagen
fibril assay. Data
are presented
as the mean 2 SEM (n = 3).
notype in culture, and their proliferation rate is not changed
in response to relaxin. However, several stromal cell activities are altered by relaxin. Theseinclude increasedgelatinase
and collagenase activities and altered glycosaminoglycan
synthesis. These relaxin-induced changes correspond to
those reported in vim late in pregnancy. Collagen fiber structure has been reported to change from dense and tightly
packed in nonpregnant and early pregnant states to loose
and randomly organized at term (6,10,12,21,54). Increases
in collagen solubility, hyaluronic acid and heparin sulfate
levels, and water content as well as decreasesin collagen,
dermatan sulfate, and chondroitin sulfate concentrations
have been reported during late pregnancy (5, 7-14). In addition, increasesin collagenolytic and other proteolytic activities were observed in viva (6). There is a consensusthat
the enzymatic breakdown of collagen through the actions of
collagenasesand proteinases constitutes in part the cellular
basisof cervical softening before labor. The cervical structure
is further weakened by the increasesin hyaluronic acid and
water content that are major factors in the soft swollen appearance of the cervix at term. In the gilt, relaxin has been
JCE & M.1996
Vol81.No9
ET AL.
=(VD
200
6
"0
=.
150
IIr
E
z
100
8
3
yl
50
I
n
0
0
1
Relaxin
I
10
100
(rig/ml)
FIG. 5. Effects
of relaxin
on [3Hlglucosamine
incorporation
by cultured human
cervical
stromal
cells. Cultures
were treated
with the
indicated
dose of porcine relaxin
for 7 days, and 13Hlglucosamine
was
added for the last day of relaxin treatment.
Glycosaminoglycans
in the
cell (counts per min x 10) and secreted
into the medium
(counts per
min X 100) were isolated
as described
in Materials and Methods. Data
are presented
as the mean 2 SEM (n = 3).
shown to increase cervical weight and softening, changes
associated with increased cervical matrix material and increased cervical hydration (27, 28). Furthermore, these
changes in cervical ripening are blocked by the concomitant
administration of antibody specific for relaxin (24). Cellular
hyperplasia is not induced by relaxin in these animals, and
the weight gain is due primarily to the increase in the acellular matrix. Relaxin has profound effects on cervical collagen fibers (28); morphometric analysis has shown a decreased ratio of collagen to amorphous ground substance
that directly correlates with the extent of cervical softening
(55). In the gilt as in human cervical cell cultures, the effects
of relaxin do not to appear to be dependent upon prior
estrogen exposure. This is different from what was found in
some rodent models, in which steroids potentiate the effect
of relaxin (16, 26).
There is no indication of significant increasein cervical cell
proliferation after relaxin treatment in the gilt. The relaxininduced increase in cervical weight has been attributed to
increased tissuehydration and increasedextracellular matrix
(27). Similarly, no enhanced cell proliferation was found in
the cultured human cervical stromal cells in vitro. We found
in cervical stromal cell cultures a relaxin-induced increasein
the synthesis of cellular and secreted glycosaminoglycans,
which is an important component of the stromal extracellular
matrix.
Although it has been recognized for many years that the
cervix is a source of metalloproteinases, the identity of these
proteinases have yet to be defined. Several metalloproteinase,with low molecular masses,such as the relaxin-induced
36-kDa activity reported here, have been described. These
include PUMP-1 (56), a gelatinase present in l-day postpartum rat uteri but absent at 5 days postpartum; a 28-kDa form
of human stromelysin (57); and a 24-kDa rabbit stromelysin
RELAXIN
MODULATES
CERVICAL
(58) that functions as a proteoglycanase / procollagenase activator. Furthermore, in response to relaxin, human dermal
fibroblasts secrete a collagenase that migrates as a 57/ 52-kDa
doublet (59). We have not determined whether the doublet
found in our cervical cells corresponds to this collagenase.
There is limited information available regarding the effects
of relaxin on glycosaminoglycan
distribution in the cervix. It
has been reported that porcine relaxin increases hyaluronic
acid and heparin sulfate content in the cervix of steroidtreated ovariectomized pregnant rats (23). We have examined glycosaminoglycan
synthesis in cultured human cervical stromal cells after relaxin administration.
Although we
demonstrate a relaxin-induced increase in glycosaminoglycan synthesis, we have not characterized these products.
This study is the first to demonstrate that relaxin directly
modulates the activity of human cervical cells in a manner
consistent with its effects in vim. Furthermore, these effects
are not dependent upon prior steroid exposure. These studies clearly demonstrate the importance of research aimed at
understanding the cellular and molecular basis for the effects
of relaxin on the human cervix and the need for an understanding its effects on individual cell types of the cervix.
Relaxin has recently been shown to bind to cervical epithelial,
stromal, and smooth muscle cells (60-62). Therefore, the
control of cervical tissue biology by relaxin probably results
from the coordinate control of a number of different cervical
cell types. These studies need to be extended to the human
model if an effective cervical ripening protocol is to be
developed.
STROMAL
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Acknowledgments
The authors thank the National
Hormone
and Pituitary
Program
for
the porcine relaxin,
Mr. Lenon Dong for technical
assistance,
and Dr.
Eleana McCoy
for suggestions
regarding
the manuscript.
25.
26.
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