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: mbogusiewicz@yahoo.com

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.

544 M. Bogusiewicz et al.

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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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ssel

dorf

on

04/0

9/13

For

pers

onal

use

onl

y.