UTERY
Activity of matrix metalloproteinase-2 and -9 and contents of theirtissue inhibitors in uterine leiomyoma and corresponding myometrium
MICHAŁ BOGUSIEWICZ1, MARTA STRYJECKA-ZIMMER2, KRZYSZTOF POSTAWSKI1,
ARTUR J. JAKIMIUK1,3, & TOMASZ RECHBERGER1
1Second Department of Gynecology, Medical University of Lublin, Lubin, Poland, 2Department of Biochemistry and
Molecular Biology, Medical University of Lublin, Lubin, Poland, and 3Polish Academy of Science, Medical Research Centre,
Warsaw, Poland
(Received 2 August 2006; revised 4 July 2007; accepted 6 July 2007)
AbstractBackground and aim. Matrix metalloproteinase-2 and -9 (MMP-2 and -9) are proteolytic enzymes degrading extracellularmatrix proteins, mainly collagen type IV. Recent reports show that these proteases may be implicated in the growth of uterineleiomyoma. The aim of the present study was to evaluate the activity of MMP-2 and MMP-9, the contents of their tissueinhibitors (TIMP-1 and TIMP-2) and the immunolocalization of collagen type IV in uterine leiomyoma and correspondingmyometrium.Materials and methods. Material for the study comprised specimens of uterine leiomyomas and corresponding myometriumderived from 20 hysterectomized women. The activity of MMP-2 and MMP-9 in tissue extracts was evaluated by semi-quantitative zymography. TIMPs were measured by enzyme-linked inmmunosorbent assay. Protein immunohistochemistrywas applied for detection of collagen type IV.Results. Activity and activation ratio of MMP-2 were significantly higher in leiomyomas than myometrium. The activity ofMMP-9 was weak and did not differ between the investigated tissues. Contents of TIPM-1 and TIPM-2 were similar in bothtissues. In both leiomyomas and myometrium, collagen type IV was localized in the extracellular matrix embedding bundlesof smooth muscle cells, but was absent in areas of extracellular matrix accumulation within leiomyomas and in larger septaseparating muscle fibers in normal myometrium.Conclusion. MMP-2 may be implicated in pathogenesis of leiomyoma.
Keywords: Uterine leiomyoma, metalloproteinase, collagen
Introduction
Uterine leiomyomas (fibroids) are the most common
tumors of the female pelvis. Despite its high pre-
valence, the pathophysiology of this benign neoplasm
is still not completely understood. It is well docu-
mented that leiomyomas arise from a single smooth
muscle cell (SMC) of the uterus and subsequently
grow by clonal expansion [1,2]. Their development is
affected by ovarian hormones, both estrogens and
progestins [1,3]. Recent studies emphasize an im-
portant role of the interplay between cytokines,
growth factors and extracellular matrix (ECM)
components in the pathogenesis of uterine leio-
myoma [4].
More than half of the leiomyoma dry tissue mass is
composed of ECM [5,6]. The content of ECM is
markedly higher in fibroids than in corresponding
myometrium, with collagen type I being the most
prevalent extracellular protein [5]. Recently, it has
been shown that growth of uterine leiomyomas may
be related to increased activity of matrix metallopro-
teinase-2 (MMP-2) [7], a proteolytic enzyme effec-
tively degrading collagen type IV as well as collagen
types I, V, VII, X and XI, gelatine, elastic, fibronectin,
laminin, entactin and proteoglycans [8–10]. MMP-2
and MMP-9 (which is also capable of degrading
collagen type IV) belong to a family of extracellular
endopeptidases – the matrix metalloproteinases
(MMPs). Besides the ability to cleave ECM proteins,
Correspondence: M. Bogusiewicz, Second Department of Gynecology, Medical University of Lublin, Jaczewskiego 8, 20-954 Lublin, Poland.
Tel: 48 81 7244686. Fax: 48 81 7244849. E-mail: [email protected]
Gynecological Endocrinology, September 2007; 23(9): 541–546
ISSN 0951-3590 print/ISSN 1473-0766 online ª 2007 Informa UK Ltd.
DOI: 10.1080/09513590701557416
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these enzymes are distinguished by the presence of
a zinc ion at the active site, secretion as latent
zymogens with subsequent activation associated
with the loss of a *10-kDa propetide, and inhibi-
tion by tissue inhibitors of metalloproteinases
(TIMPs) [11].
Dou and co-workers [12] reported that MMP-2
and MMP-9 as well as their inhibitors are produced in
both fibroids and healthy myometrium. Expression of
MMP as well as TIMP mRNAs was higher in normal
tissue compared with tumors. After treatment with
gonadotropin-releasing hormone (GnRH) agonist
expression of metalloproteinases increased whereas
expression of their inhibitors decreased in both
tissues, suggesting that metalloproteinases participate
in the process of leiomyoma regression. However,
Wolanska and colleagues [7] showed that the activity
of MMP-2 is higher in large (weighing 4100 g)
leiomyomas but lower in smaller (weighing 510 g)
tumors compared with myometrium taken from
normal uterus. An immunohistochemical examina-
tion revealed MMP-2 protein in only two out of 26
leiomyomas [13].
Since the role of MMP-2 and MMP-9 in the
pathogenesis of uterine fibroids is still unclear, in the
present study we aimed to evaluate the activity of these
enzymes in uterine leiomyoma and corresponding
normal myometrium after applying an extraction
method which enables retrieval of MMPs from
ECM. Furthermore, we attempted to measure the
contents of their tissue inhibitors, TIMP-1 and
TIMP-2. Because collagen type IV appears to be the
main substrate for the metallproteinases investigated,
immunolocalization of this protein within leiomyo-
mas and myometrium was also checked.
Methods
Material
Materials for the study were paired specimens of
uterine leiomyoma and corresponding normal myo-
metrium derived from 20 premenopausal women
aged 45–55 years (mean: 49.2 years) who underwent
total abdominal hysterectomy. None of them was
previously treated with GnRH analogs or other
hormonal agents. The specimens were taken im-
mediately after removal of the uterus, frozen in liquid
nitrogen and stored at 7758C until further proces-
sing. The size of tumors ranged from 2 to 5 cm.
Three tumors were submucosal, 13 intramural and
four subserosal. Leiomyomas presenting with any
kind of degenerative changes were not included.
Follicular or luteal phase of the menstrual cycle was
evaluated by the day of the cycle and histological
appearance of the endometrium. In 11 cases speci-
mens were taken during the luteal phase and in nine
cases during the proliferative phase.
The study was approved by the Ethical Committee
of the Medical University of Lublin.
Extraction of metalloproteinases
Metalloproteinases and their inhibitors were ex-
tracted using the method described by Woessner
[14]. Briefly, tissue specimens were immersed in
liquid nitrogen, pulverized and homogenized in
20 volumes of cold 0.25% Triton X-100 dissolved
in 10 mM CaCl2. After centrifugation at 6000g for
30 min at 48C, the supernatants (Triton extracts)
were obtained. The remaining pellets were re-sus-
pended in 20 volumes of 50 mM Tris buffer, pH 7.4,
containing 100 mM CaCl2 and 150 mM NaCl. The
samples were then incubated at 608C for 6 min with
constant agitation and next centrifuged at 20 000g
for 30 min at 48C. The resulting supernatants (heat
extracts) were recovered.
The extracts were dialyzed overnight against
50 mM Tris buffer, pH 7.5, containing 10 mM
CaCl2, 200 mM NaCl, 0.02% NaN3 and 0.05%
Brij-35. Lowry’s method with the modification of
Cadman and associates [15] was used for evaluation
of the protein contents of the extracts.
Evaluation of activity of matrix metalloproteinase-2
and -9 by zymography
Identification of MMP-2 and MMP-9 and assess-
ment of their activities were performed with zymo-
graphy based on the method described by Hibbs and
collaborators [16]. In this method, being a type of
substrate electrophoresis, MMP-2 and MMP-9
degrade gelatine incorporated into gels that results
in negative (unstained) bands. It enables detection of
active and latent forms of gelatinases at proper mole-
cular weight positions.
Zymography was performed in 10% sodium
dodecyl sulfate–polyacrylamide gels, 0.75 mm thick,
containing Type A Porcine Skin Gelatin, 300 Bloom
(0.8 mg/ml, G2500; Sigma, St. Louis, MO, USA).
Extracts (5 mg protein per lane) were electrophoresed
at 25 mA constant current, in a cold room, using a
Mini-PROTEAN II apparatus (BioRad, Richmond,
CA, USA). Then the gels were washed with 2.5%
Triton X-100 two times for 15 min each and another
two times for 5 min each with 50 mM Tris buffer,
pH 7.6, containing 10 mM CaCl2, 5 mM ZnCl2, 1%
Triton X-100 and 0.02% NaN3. Subsequently, the
gels were incubated in 50 mM Tris buffer, pH 7.6,
containing 10 mM CaCl2, 5 mM ZnCl2, 1% Triton
X-100 and 0.02% NaN3, for 18 h at 378C with
aminophenylmercuric acetate (an activator of
MMPs) at the final concentration of 1 mM. The
gel slabs were stained with 0.1% Coomassie Brilliant
Blue R-250 in 50% methanol and 7% acetic acid for
3 h, and destained in 7% acetic acid for 1 h.
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In additional experiments, a specific inhibitor of
metalloproteinases (i.e. 1,10-phenanthroline; P 9375,
Sigma) and a serine proteases inhibitor (i.e. phenyl-
methylsulfonyl fluoride; P 7626; Sigma) were applied
to confirm that digestion of gelatine was caused by the
MMPs.
Data analysis
The gels were scanned in grey-scale mode at 600 dpi
using iPhotoPlus software, version 1.2 (Ulead Sys-
tems, Inc., Taipei, Taiwan). Images were inverted,
bands identified and integrated optical density (OD)
calculated using ONE-Dscan version 3.1 (Scanaly-
tisc, Inc., Fairfax, VA, USA). Since in a pilot study a
linear correlation between integrated OD multiplied
by lane width and activity of standard MMP-2
(catalog no. 1782916; Roche Diagnostics, Basel,
Switzerland) was observed, these parameters were
used for quantification of MMP activities by compar-
ison of the bands detected in investigated samples
with bands produced by 10 mU of standard MMP-2,
which was run on each gel. Activities of MMP-2 and
MMP-9 are displayed as arbitrary units (U). One
arbitrary unit is defined as the gelatinolytic activity of
10 mU of standard MMP-2 in given conditions. All
values were normalized per gram of protein.
Evaluation of content of tissue inhibitor of
metalloproteinase-1 and -2
Concentrations of TIMP-1 and TIMP-2 were
measured by enzyme-linked immunosorbent assay
(Biotrak ELISA systems RPN2611 and RPN2618;
Amersham Pharmacia Biotech, Uppsala, Sweden).
For evaluation of TIMPs mixtures of Triton extracts
and heat extracts with equal protein contents were
prepared. The assay for TIMP-1 recognizes free
inhibitor as well as that complexed with metallopro-
teinases. In the case of TIMP-2 the assay identifies
free inhibitor as well as inhibitor complexed with
active enzymes but not with latent MMP-2.
Immunohistochemical analysis of collagen type IV
expression
Immunohistochemical analysis of collagen type IV
protein expression was performed using the avidin–
biotin–peroxidase complex method with the purified
mouse anti-human collagen type IV AB-2 mono-
clonal antibodies (catalog no. CP 56; Oncogene, San
Diego, CA, USA). After routine deparaffinization
and rehydration, antigen retrieval was accomplished
by microwaving sections in citrate buffer (pH 6.0).
The sections were then washed in phosphate-buffered
saline (PBS), pH 7.6, and incubated with normal
serum as the blocking reagent to minimize non-
specific binding. The primary antibody was applied
overnight at a dilution of 1:100 and sequentially
followed by biotinylated anti-mouse immunoglobulin
and horseradish peroxidase-conjugated streptavidin
(ABC-Vector Laboratories, Burlingame, CA, USA).
Sections were then incubated in the chromogenic
substrate solution of PBS with 0.1% DAB (3,30-diaminobenzidine tetrahydrochloride) and 0.02%
H2O2 and 0.0068% imidazol for 5–7 min. Finally,
sections were counterstained with Harris’s hema-
toxylin, dehydrated, cleared and mounted.
Statistical analysis
Statistical analysis was performed with the Statistica
software package, version 5 (StatSoft Inc., Tulsa, OK,
USA), utilizing Student’s t tests for dependent and
independent samples, the Mann–Whitney U test and
Wilcoxon’s test when appropriate. A value of p5 0.05
was considered as statistically significant. Results are
presented as mean+ standard error of the mean.
Results
Zymography revealed activity of both latent (72 kDa)
and active (62 kDa) MMP-2 in all investigated
samples (Figure 1). MMP-2 activity was significantly
higher in leiomyomas than in normal myometrium
(Figure 2). Active/latent MMP-2 ratio was also
Figure 2. Activity of matrix metalloproteinase-2 (MMP-2) in
leiomyoma and normal myometrium evaluated by zymography.
Data are means, with standard error of the mean shown by vertical
bars. Significantly higher activity of MMP-2 was found in
leiomyoma vs. myometrium (p5 0.001).
Figure 1. Analysis of the activity of matrix metalloproteinase
(MMP)-2 and -9 by zymography in leiomyoma (L) and myome-
trium (M). Negative bands are caused by digestion of gelatine. The
electrophoretic positions of latent (92 kDa) MMP-9, latent
(72 kDa) MMP-2 and active (62 kDa) MMP-2 are indicated.
Activity MMP-2 and MMP-9 in uterine leiomyoma 543
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higher in leiomyomas than myometrium (0.56+0.05 vs. 0.27+ 0.04, p5 0.001). Most of the total
MMP-2 activity, i.e. 88.5% in leiomyomas and
85.9% in myometrium, was detected in heat
extracts. Higher MMP-2 activity in leiomyomas than
in myometrium was also observed when data were
compared separately for the heat extracts and Triton
extracts.
In contrast to MMP-2, MMP-9 exhibited weak or
undetectable activity. In none of the examined tissues
was the active form of MMP-9 found (Figure 1).
Higher MMP-9 activity was found in heat extracts
(69.9% of total activity in leiomyomas and 64.5% in
myometrium).
The activity of neither MMP-2 nor MMP-9
differed significantly between fibroids of different
size or location. Both in leiomyomas and myome-
trium, the gelatinolytic activity as well as the active/
latent MMP-2 ratio were similar in proliferative and
luteal phases of the menstrual cycle.
Contents of TIMP-1 and TIMP-2 did not
differ significantly between the investigated groups
(Table I). No differences in contents of inhibitors
were observed between proliferative and luteal
phases of the menstrual cycle. Size or localization of
tumors did not influence the concentration of
inhibitors. Interestingly, in leiomyomas, a negative
correlation between TIMP-2 content and MMP-2
activity was observed (r¼70.78, p5 0.05). We did
not find such an association in normal myometrium.
Immunohistochemical analysis for collagen type
IV showed positive staining of similar intensity both
in leiomyomas and myometrium (Figure 3). In
leiomyomas collagen type IV was localized in the
ECM embedding bundles of SMCs, whereas areas of
ECM accumulation were negative for this protein.
Similarly, in myometrial samples collagen type IV
surrounded SCM bundles, but did not contribute to
the formation of larger septa which separate muscle
fibers.
Discussion
MMP-2 is a matrix metalloproteinase expressed
constitutively in a number of tissues. Many physio-
logical and pathological processes are associated with
increased activity of this enzyme [8–11]. Our results
suggest that MMP-2 may also play a role in the
pathogenesis of uterine leiomyoma.
Growth of leiomyomas involves two parallel
processes: proliferation of SMCs and deposition of
Table I. Content of tissue inhibitor of metalloproteinase (TIMP)-1
and -2 in uterine leiomyomas and myometrium.
Myometrium
(mg/g protein)
Leiomyoma
(mg/g protein)
TIMP-1 20.1+4.1 17.5+5.6
TIMP-2 28.3+9.1 27.8+3.9
Data are presented as mean+ standard error of the mean; the
differences were not statistically significant.
Figure 3. Immunohistochemical staining for collagen type IV in
human leiomyoma and normal myometrium. In leiomyoma
collagen type IV embeds bundles of smooth muscle cells (A),
whereas it is absent in areas of extracellular matrix accumulation
(arrow) (B). In normal myometrium bundles of smooth muscle cells
are surrounded by collagen type IV, which is not present in larger
septa separating muscle fibers (arrow) (C). Magnification 106.
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extracellular proteins. Both these events, which
require considerable remodeling of ECM, may
possibly involve MMP-2. As shown in Figure 3,
collagen type IV evenly encloses bundles of SMCs
within leiomyomas and myometrium. Such locali-
zation indicates that this protein, by its close
interaction with the cellular membrane, may influ-
ence the metabolism of SMCs. Cleavage of collagen
type IV and other extracellular components accom-
plished by MMP-2, resulting in dissolution of the
ECM framework surrounding cells, may lead to
modulation of cell–cell and cell–matrix interactions,
thus affecting cell differentiation and proliferation.
This hypothesis is supported by data derived from
studies on vascular SMCs, which indicate that the
ECM plays a crucial role in regulation of SMC
phenotype [17].
Furthermore, MMP-2 interfaces with certain
growth factors such as transforming growth factor-b(TGF-b) and insulin-like growth factors (IGFs),
which potentially promote cell proliferation and
ECM deposition within uterine leiomyomas [4].
TGF-b is processed into an active form by proteolytic
degradation accomplished by MMP-2 and MMP-9
[18]. MMP-2 also cleaves insulin-like growth factor
binding protein-3, an inhibitor of IGFs [19].
It is unclear if the activity of MMP-2 in uterine
leiomyoma is regulated by ovarian steroids. The
promoter regions of the MMP-2 gene do not contain
AP-1 sites [8–10]. However, Wingrove and collea-
gues [20] showed that estradiol upregulates secretion
of this enzyme by cultured human vascular smooth
cells. Conversely, expression of MMP-2 in endome-
trium is relatively constant and both estrogens and
progesterone seem to have little in vivo and in vitro
effect on its synthesis [8]. Dou’s group [12] observed
higher expression of MMP-2 mRNA in uterine
fibroids and myometrium in secretory compared
with the proliferative phase of the cycle, together with
similar immunostaining intensity of MMP-2 protein.
In our study, both in leiomyoma and myometrium,
the activity of MMP-2 did not differ between
proliferative or secretory phase. Taken together,
ovarian hormones may have some effect on expres-
sion of MMP-2 gene in fibroids and myometrium but
seem not to influence contents of MMP-2 protein
and its proteolytic activity.
MMP-2 and MMP-9, like most metalloprotei-
nases, are secreted into the extracellular space as
inactive zymogens where they undergo activation and
exert proteolytic activity [8–11]. In the present study
we applied an extraction method which enables
recovery of about 95% of metalloproteinases [14].
Incubation in 608C causes shrinkage of collagen
fibers and together with a high concentration of
calcium ions facilitates dissociation of MMPs from
ECM components. In consequence, the pool of
metalloproteinases bound in vivo to ECM is retrieved
during heat extraction [14]. In our study more than
85% of MMP-2 activity was detected in heat extracts,
indicating that both in leiomyoma and myometrium
the main pool of this enzyme is present extra-
cellulary. This finding may explain why MMP-2
immunostaining is rarely observed in uterine leio-
myoma cells [13].
In contrast to MMP-2, the activity of MMP-9 was
weak and not consistently detected in both leio-
myoma and myometrium. It indicates that this
metalloproteinase does not play a significant role in
ECM turnover within uterine leiomyomas.
Although our results regarding MMP-2 and
MMP-9 activity are generally consistent with ob-
servations reported by Wolanska’s group [7], we
believe that the methods applied in our study yielded
more reliable results. First of all, we paired samples
of leiomyomas and normal myometrium by taking
them from the same uterus to avoid variations in
hormonal milieu associated with the phase of the
menstrual cycle or the patients’ menopausal status.
These variables could bias Wolanska’s study [7], in
which control tissue was derived from other indivi-
duals who, on average, were 9 years older. Further-
more, the far more efficient extraction method used
in our study (more than 85% of MMP-2 activity was
detected in heat extracts) gave us the opportunity to
assess the activity of most the enzyme present in the
investigated samples. Wolanska and co-workers [7]
showed that activity of MMP-2 in small fibroids (less
than 10 g) was lower, whereas that in large tumors
(more than 100 g) was higher, in comparison with
control tissue. As we found no association between
tumor size and MMP-2 activity, our results do not
indicate that MMP-2 activity increases together with
leiomyoma growth.
The activity of metalloproteinases is regulated by a
group of 20–30-kDa proteins named tissue inhibitors
of metalloproteinases (TIMPs), which form non-
covalent complexes with MMPs [8–10]. Activity of
MMP-9 is preferentially inhibited by TIMP-1,
whereas TIMP-2 reveals high affinity to MMP-2
[8,10]. Interestingly, despite being an inhibitor,
TIMP-2 plays a crucial role in activation of MMP-
2. It attaches MMP-2 to membrane-type 1 matrix
metalloproteinase (MT1-MMP) anchored to cell
surface, which in turn cleaves the propeptide domain
of latent MMP-2 thus accomplishing activation of
the proenzyme [9,10]. It has been shown that,
depending on its concentration, TIMP-2 act as an
inhibitor or activator; in high concentrations TIMP-2
blocks the activity of MMP-2 [21].
In our study the contents of both inhibitors did not
differ between leiomyoma and myometrium. In
leiomyomas we observed a negative correlation
between MMP-2 activity and content of TIMP-2.
Since the antibody used for TIMP-2 detection does
not recognize inhibitor bound to metalloproteinases,
Activity MMP-2 and MMP-9 in uterine leiomyoma 545
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this finding most probably reflects engagement of
TIMP-2 in activation processes. The phase of the
menstrual cycle and, in the case of leiomyomas, size
or localization of the tumor had no effect on con-
centration of the inhibitors.
In conclusion, our study showed higher activity as
well as activation rate of MMP-2 in uterine leiomyo-
ma compared with normal myometrium, indicating
that this enzyme may be implicated in the patho-
genesis of leiomyoma. We found no differences in
MMP-9 activity and contents of TIMPs between the
investigated tissues.
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dorf
on
04/0
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For
pers
onal
use
onl
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