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In Vivo Evidence for Active Matrix Metalloproteinases in Human Endometrium Supports their Role in Tissue Breakdown at Menstruation

Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Lois A Salamonsen, Associate Professor, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: . lois.salamonsen{at}med.monash.edu.au

Abstract

Human endometrium remodels extensively during each reproductive cycle culminating in loss of most functionalis tissue at menstruation. Evidence suggests that menstruation results from the action of the matrix metalloproteinases (MMP), enzymes secreted in latent forms. MMP activation is thus an important regulatory step. It has not been established that MMPs are active within menstrual endometrium in vivo. We used in situ zymography to demonstrate active forms of MMPs in human endometrium across the normal menstrual cycle. Both gelatinase and collagenase activities were detected in most endometrial tissues. Semiquantitation demonstrated a substantial and significant increase in both gelatinase and collagenase activity in menstrual samples compared with those at any other time of the cycle. Gelatinase activity was both associated with cells and extracellular. All collagenase activity was extracellular. Immunoreactive MMP-2 and MMP-9 colocalized with active gelatinase, although much immunoreactive gelatinase was inactive. Some gelatinase activity colocalized with CD45⁺ leukocytes. Menstruation is initiated at discrete foci, and active MMPs were similarly at foci within the tissue. This is the first in vivo evidence for increased active MMPs in menstrual endometrium compared with other stages of the cycle. These findings position the MMPs for a critical role in the matrix degradation at menstruation.

THE HUMAN ENDOMETRIUM undergoes extensive cyclical remodeling during each menstrual cycle, driven by the changing milieu of female steroid hormones, estrogen, and circulating progesterone. In the absence of an implanting blastocyst, progesterone production ceases and the ensuing fall in progesterone results in the breakdown and shedding of much of the functionalis layer of the endometrium at menstruation. Many of the cells in the menstrual fluid are still living as evidenced by their potential to grow both in culture or in vivo, as seen in endometriosis (1). Thus, the tissue loss is not due merely to ischemia or cellular apoptosis but results from matrix degradation and release of tissue rafts along with individual or clustered cells. Importantly, at any one point in time during menstruation, endometrial breakdown is occurring only at discrete foci, whereas in other areas, regrowth is already in progress (2).

A considerable body of evidence now supports an important role for the matrix metalloproteinases (MMPs) in the process of menstruation. MMPs are a family of enzymes that together have the capacity to degrade all components of the extracellular matrix, both interstitial matrix to which the endometrial stromal cells form attachments, and basal lamina, which underlies endometrial glandular and luminal epithelium and blood vessels and which surrounds individual predecidual cells late in the menstrual cycle. Importantly, MMPs are produced in latent forms and a critical level of regulation is their activation by other proteases. Endometrial MMP expression is markedly increased at menstruation (3, 4, 5). The individual MMPs that appear to be of particular relevance are MMP-1 (interstitial collagenase), MMP-3 (stromelysin 1), MMP-2 and MMP-9 (gelatinases A and B, respectively) and membrane-type 1 MMP. A major focus of recent work has been on the regulation of MMPs by endometrial explants or by separated endometrial cell types, and it is apparent that withdrawal of progesterone (5, 6, 7, 8) or stimulation by a range of paracrine regulators produced by endometrial epithelial (9), and stromal cells (9, 10) or by endometrial leukocytes (11, 12) can regulate endometrial MMP expression and activation in vitro. However, endometrium also expresses substantial quantities of tissue inhibitors of metalloproteinases (TIMP)-1, -2, and -3 (13) that can inactivate MMPs by binding their active forms with a 1:1 stoichiometry. Thus, for matrix breakdown, there must be a change in the balance between active forms of MMPs and TIMPs within the microenvironment.

The MMPs are produced and secreted as latent forms and to date, antibodies are not available that detect only active forms of the enzymes. Thus, neither gene expression studies nor immunologically based techniques provide evidence for active forms of MMPs. It is also not possible to establish whether any MMP detected is TIMP bound by such means. In vitro studies have provided a measure of support for MMP activation within the endometrium. Explants of endometrium undergo breakdown in culture in the absence of steroid hormones and this can be reversed by inhibitors of MMPs (14). Furthermore, substrate zymography has shown active forms of MMPs in extracts of menstrual endometrium (15, 16). The presence of MMP activity within endometrial tissue in an in vivo situation has not yet been demonstrated; neither has it been shown that such activity is enhanced within menstrual endometrium.

This study demonstrates for the first time, the presence of biologically active forms of MMPs in snap-frozen samples of human endometrium, by application of the technique of in situ zymography (17, 18). Both gelatinase and collagenase activities were present at discrete foci in endometrial tissues and a significant increase in these activities was measured during menstruation. The activities were inhibited by a metalloprotease inhibitor, and in some instances, colocalized with markers for leukocytes. Both MMP-2 and MMP-9 were identified in their active forms. These data provide substantial support for a critical role for MMPs at menstruation.

Materials and Methods

Tissue collection and patient details

Endometrial biopsies were collected at dilatation and curettage during minor gynaecological investigations of fertile women reporting normal menstrual cycles and with no history of endometrial pathology. The stage of the menstrual cycle was confirmed by histological dating according to previously described criteria (19); thereafter, tissues were allocated to one of seven groups, menstrual (d 1–4, n = 3), early, mid, and late proliferative (d 5–8, 9–12 and 13–14, n = 3, 4, 6, respectively) and early (d 15–18, n = 4), mid (d 19–23, n = 6) and late (d 24–28, n = 4) secretory phases. All tissue collections were approved by the Institutional Human Ethics Committee and written informed consent was obtained from all women participating in the study. Endometrial samples were snap frozen immediately after collection in OCT compound (Sakura Finetek, CA) and stored at -80 C for no more than 2 wk before use.

In situ zymography

Substrates for in situ zymography were: DQ gelatin from pig skin, fluorescein conjugate and DQ collagen type I from bovine skin, fluorescein conjugate (Molecular Probes, Inc., Eugene, OR).

Twenty-four 7-µm sections were cut from the center of each tissue block on to poly-L-lysine-coated slides. The slides were then kept at 4 C. Of every six serial sections cut, the first was subjected to collagen zymography, the fourth to gelatin zymography, and adjacent sections to hematoxylin and eosin staining. Thus, four replicate sections, separated by 35 µm, were examined for each of gelatinase and collagenase activity, for each tissue block.

For in situ zymography, the tissue section was fixed in 10% buffered formalin for 5 min at 4 C and washed three times with cold PBS (pH 7.4). Where nuclear counterstaining was required, propidium iodide (Molecular Probes, Inc.) diluted 1:50 in PBS was applied for 8 min at room temperature, and the tissue then was thoroughly washed with cold PBS. The slide was held in a darkened PBS bath at 4 C until use. The desired substrate (DQ gelatin or DQ collagen) was dissolved to a final concentration of 25 µg/ml in a mixture of 2% gelatin and 2% sucrose in PBS with 0.02% sodium azide. One hundred microliters of substrate were layered over the tissue section, covered with a coverslip and incubated in a darkened humid chamber at 37 C for 16 h. Where required, the broad-spectrum MMP inhibitor, 1,10-phenanthroline (Sigma, St. Louis, MO); final concentration 10 mM) was added to the section before counterstaining and incubated at 37 C for 1 h. Each section was viewed using an Olympus Corp. (Birkeroed, Denmark) fluorescent microscope with FITC filter, fitted with a Fuji Photo Film Co., Ltd. (Tokyo, Japan) HC-2000 high resolution digital camera and Analytical Imaging System 3.0 software (Imaging Research, Inc., St. Catherine’s, Ontario, Canada).

Semiquantitation of active MMPs

The relative quantities of active MMPs in the tissue blocks were assessed. As active MMP appeared as discrete spots of fluorescence, for each section, the number of these spots was counted and the total area of the section (mm², inclusive of all cellular compartments) was measured using an M.D.4 microscope digitizer (Accustage, Shoreview, MN) at x40 magnification. Activity was expressed as number of spots of activity per mm². Four sections from each block, taken at 35-µm intervals, were similarly treated and the mean activity for each block calculated. The data were then combined for all the blocks from each phase of the menstrual cycle. Sections for semiquantitation were not counterstained as this resulted in some quenching of the fluorescence arising from the cleavage of substrate. Data were subjected to ANOVA and Tukey’s post hoc test and significant differences between groups taken as P < 0.05.

Colocalization studies

Due to the lengthy staining protocols for MMP-2 and MMP-9, during which enzyme activity was lost, it was not possible to perform both in situ zymography and immunostaining on the same section. Therefore, serial 3 µm sections were cut and adjacent sections subjected separately to the different protocols. MMP-2 was immunolocalized using the mouse antihuman MMP-2 antibody, clone 42–5D11 (Calbiochem, San Diego, CA) and the A568 amplification kit (Molecular Probes, Inc.) with Texas Red as the fluorochrome. MMP-9 staining used the mouse antihuman MMP-9 antibody, clone 56–2A4 (Calbiochem) with the same detection system. The sections were fixed with 10% buffered formalin for 30–40 min at 4 C, washed in Tris-buffered saline (TBS), pH 7.4, with 0.3% Tween 20 for 10 min at room temperature, blocked with 10% normal rabbit serum/10% normal goat serum in TBS for 10 min and incubated with either anti-MMP-2 or anti-MMP-9 diluted to 2 µg/ml for 60 min at 37 C. Mouse IgG at similar protein concentration was used as the negative control. After thorough washing, the amplification kit was applied twice. Fluorescent staining was examined under a green filter. In each case, the adjacent section to that immmunostained, was subjected to gelatin substrate in situ zymography as detailed above.

Colocalization studies were carried out on single sections to determine whether MMP activity in the tissues was associated with the presence of leukocytes. In brief, the leukocytes were detected on 7 µm sections using an antiserum against leukocyte common antigen (CD45, clone 2B11+PD7126, DAKO Corp., Carpinteria, CA). The sections were fixed for 10 min with 10% formalin at 4 C, washed with TBS, and the primary antibody applied at a 1:10 dilution, for 10 min at 37 C. After a further wash with TBS, a Texas Red-linked antimouse IgG (Molecular Probes, Inc., diluted 1:10) was applied for 12 min at 37 C. After further washing, the 4'-6-diamidino-2-phenylindole, dihydrochloride nucleic acid counterstain (Molecular Probes, Inc., 200 µM) was applied for 4 min at room temperature. Following washing in TBS, the sections were layered with the substrate gel and subjected to in situ zymography as above. Leukocytes were visualized as red fluorescence, whereas active MMP was green and nuclear counterstain was blue. It is important to note that the fluorescence resulting from substrate cleavage was somewhat diminished in these dual-detection sections compared with those subjected only to in situ zymography and such sections were not used for semiquantitation.

Results

Detection of gelatinase activity

When sections of endometrial tissue were subjected to in situ zymography with a gelatin substrate, foci of fluorescence, representing gelatin degradation were observed. These foci were always within the stromal areas of the tissue and were evident in low numbers within the tissue at all stages of the menstrual cycle (Fig. 1, A–C) except the menstrual phase when they were more numerous. In counterstained sections, it was apparent that fluorescence was either cell-associated (Fig. 1D) or extracellular (Fig. 1E). In two sections where considerable decidualization of the stromal cells had occurred, gelatinase activity was also associated with the decidual cells (Fig. 1F). No fluorescence was detected when the sections were pretreated with 1,10 phenanthroline (Fig. 1, G and G') demonstrating that the degradative activity was due to metal-dependent proteases.

View larger version (121K): [in this window] [in a new window]   Figure 1. A–G, Gelatin in situ zymography on endometrial tissue from. A, Day 11; B, d 22; C–E, d 2, showing cell surface (D) and extracellular (E) location of activity, (F) decidualized tissue on d 28 (G and G') same tissue without and with preincubation with 1,10 phenanthroline. H–J', Collagen in situ zymography on endometrial tissue. H, Premenstrual tissue; I, menstrual tissue, d 2. J, J', Counterstained sections showing extracellular location. K–L, Localization of immunoreactive MMP-2 (K) and MMP-9 (L) with gelatin in situ zymography on serial sections (K' and L', respectively). Arrows demonstrate areas of identity. M–Q, Colocalization of (M) CD45 (leukocyte common antigen) and (N) active gelatinase. O, Combined fields; P, nuclear counterstain demonstrating the lobular nucleus in the stained cell. Q, Demonstrates that not all CD45 cells contain active gelatinase. Error bars shown represent 50 µm (A, B, C, G, G') or 25 µm (all other figures).

Collagenase activity was detected when the sections were overlaid with the collagen substrate; this was also seen in small foci within tissues from all phases of the cycle but areas of collagenase activity were most abundant immediately premenstrually and during the menstrual phase (Fig. 1, H and I). Although counterstaining of the sections with propidium iodide quenched some of the fluorescence, it was still possible to establish that collagenase activity is always extracellular (Fig. 1, J and J'). Fluorescence was totally lost when the sections were pretreated with 1.10 phenanthroline.

Gelatinase and collagenase activities are elevated during the menstrual phase

Given that MMP activity is present only at small foci within the endometrium, it was important to examine more than one section from each sample of tissue. We applied in situ zymography for both gelatinase and collagenase activity, to four sections from each tissue, taken at 35µm intervals. Activity for each section was expressed as number of spots of activity per mm² and for each tissue, the mean for four sections was calculated. A total of 30 different endometrial tissues were included in the study. This semiquantitation of gelatinase activity during each phase of the menstrual cycle showed that the number of sites of activity were increased 6-fold during the menstrual phase compared with other times during the menstrual cycle (P < 0.05 vs. early proliferative and late secretory, P < 0.01 vs. each other phase) (Fig. 2A). The number of sites of collagenase activity were increased 10-fold (P < 0.01) during the menstrual phase compared with all the other phases (Fig. 2B).

View larger version (12K): [in this window] [in a new window]   Figure 2. Semiquantitative data from gelatin (A) and collagen (B) substrate in situ zymography performed on endometrial samples taken across the normal menstrual cycle. Data represents the mean ± SEM number of spots of activity per mm2 of tissue, for each phase of the menstrual cycle. Men is menstrual phase; ep, mp, and lp are early, mid, and late proliferative phase; and es, ms, and ls, early, mid, and late secretory phases of the normal menstrual cycle, respectively. Numbers of subjects are given within each histogram.

To establish whether the gelatinase activity was due to the action of MMP-2 (gelatinase A) or MMP-9 (gelatinase B), adjacent sections of menstrual endometrium were stained for either immunoreactive MMP-2 or MMP-9, and examined for activity by in situ zymography. Both MMP-2 and MMP-9 colocalized with some spots of gelatinase activity (Fig. 1, K and K' and L, L', respectively). However, only a small part of the immunoreactive MMP-2 or immunoreactive MMP-9 was represented by active gelatinase, demonstrating that in the tissue, only a fraction of the enzyme that is present is biologically active at detectable levels. It is important to note that 3-µm sections were used so that single cells or foci of activity were visualized in adjacent slides. The activity thus demonstrated is of necessity less than that seen with the thicker 7-µm sections used routinely in our laboratory for in situ zymography.

Gelatinase activity is contributed by leukocyte MMPs

We have previously demonstrated that subsets of endometrial leukocytes in the endometrium produce immunoreactive MMP-9 (20, 21), and MT1-MMP (5). MMP-2 is not known to be produced by leukocytes except T cells (22) that are in very low abundance in the functionalis layer of endometrium (23). We performed colocalization of leukocytes using an antibody against the common leukocyte marker CD45 (expressed on all cells of hematopoietic origin except erythrocytes), and gelatin substrate in situ zymography to determine whether leukocyte MMPs are indeed active in this tissue. It was apparent that some but not all of the gelatinase activity in the tissue was contributed by CD45⁺ leukocytes (Fig. 1, M–P), whereas there were also leukocytes that were not associated with the presence of active gelatinase (Fig. 1Q). The exact nature of the leukocytes was not examined: they have previously been shown to include neutrophils, eosinophils, endometrial natural killer cells, and macrophages (23).

Discussion

This study reports for the first time that MMPs are present in biologically active forms in human endometrial tissue in vivo and that the amount of active MMP, both gelatinase and collagenase, is substantially and significantly increased at menstruation. Importantly, it localizes active MMPs to discrete foci within the stroma, consistent with the focal breakdown of endometrium at menstruation. The MMP activity is both cell associated and extracellular, and in some cases, colocalizes with leukocytes, which are abundant in menstrual endometrium (23). Gelatinase activity was also detected in areas of the stroma where extensive decidualization had occurred. The activity could be inhibited by the application of metalloprotease inhibitors but not by inhibitors of other classes of proteases. The gelatinase activity colocalized with both immunoreactive MMP-2 and MMP-9. Because active MMP is detectable in menstrual endometrium, it follows that TIMP activity is not sufficient in amount and location to block the proteinase activity. These data lend support to the contention that MMPs contribute to the extensive tissue destruction at menstruation.

The human endometrium is a very unusual adult tissue, in that it undergoes constant cyclical remodeling during a woman’s reproductive life. This includes laying down of matrix, proliferation of cells, and angiogenesis during the proliferative phase of the cycle, when estrogen is the dominant ovarian steroid hormone, and a range of differentiative processes, driven by progesterone, during the secretory phase of the cycle. It is to be expected that such remodeling events would be accompanied by limited action of MMPs, given their diversity of roles, not only in degradation of matrix molecules, but also in the release and activation of biologically active molecules, such as growth factors and vasoactive substances, and in the regulation of processes such as apoptosis and cell migration (24). The finding of some small foci of MMP activity in the endometrium at all stages of the cycle, is consistent with this notion.

This study has specifically examined the activity of the gelatinases and collagenases in the endometrium. There are two gelatinases, gelatinase A (MMP-2) and gelatinase B (MMP-9) and 3 collagenases (MMP-1, MMP-8, and MMP-13) (25). The membrane type (MT)-MMPs (MMP-14 to -17) also display collagenolytic activity (26). Of these, MMP-2, MMP-9, MT1-MMP and MMP-1 have been immunolocalized in the endometrium, and there is considerable variation in their cellular source within the tissue. It is important to note that the antibodies used in such studies are not able to distinguish between latent and active forms of each enzyme. MMP-2 is the most widely expressed reflecting its constitutive production by cells. It is detected in most endometrial cells (epithelial, stromal, vascular, but not leukocytes) but with greatest intensity in degrading menstrual tissue. Its activator, MT 1-MMP is also present throughout the cycle, is patchy in epithelium and decidual cells and very strongly stained in subsets of macrophages, neutrophils, and endometrial natural killer cells but not mast cells or eosinophils (5). By contrast, MMP-9 is found in epithelium only during the early secretory phase, and at menstruation is predominantly present in a variety of leukocytes (20). MMP-1 is a stromal cell product, is barely detectable throughout most of the cycle, but immediately before and during menstruation, it is located extracellularly in small foci (27), often associated with areas of matrix breakdown (28). MMP-8 is a neutrophil enzyme and neutrophils comprise up to 15% of the total cells in the endometrial stroma immediately before menstruation (29). In the present study, no MMP activity was detected within the epithelium. Collagenase activity was clearly extracellular, and closely reflected the immunolocalization pattern reported previously for MMP-1 (27), although the possibility of activated neutrophils as the cellular source cannot be discounted. Gelatinase activity colocalized with both MMP-2 and MMP-9 immunoreactivity. It is important to note that only a small proportion of the immunoreactive material coincided with substrate degradation and that there is therefore considerably more latent than active gelatinase present in the tissue. This is consistent with data from zymography of tissue extracts where progelatinases are far more abundant than active enzymes (30). It must also be remembered that for MMP bioactivity, the active MMP must be present in excess over TIMPs within the tissue microenvironment. At least some of the gelatinase activity colocalized with CD45, supporting a contribution of MMP-9 from leukocytes. Some gelatinase activity was localized on the surface of cells and is most likely bound to MT1-MMP, which activates MMP-2 at foci on cell surfaces (31). Given the extracellular nature of much of the activity, it was not possible to determine the proportion contributed by leukocytes compared with stromal cells.

Zymographic analysis of homogenized endometrial tissue from women at different phases of the menstrual cycle has previously shown that, whereas active MMP-2 is detectable throughout the cycle, active MMP-9 is increased before and during menstruation (16, 30). In cycling women over 40 yr, the amount of active MMP-2 was increased, reflecting the increased potential for bleeding in perimenopausal women (16). It will be important to establish by in situ zymography that there is increased active MMP in the endometrium of older cycling women as demonstrated here at menstruation.

In all studies demonstrating active MMPs in menstrual endometrium (14, 30, 32), manipulation of the tissues occurs following their removal presenting the possibility that spurious activation of MMPs could occur. Indeed, cytokine production and leukocyte activation can be stimulated in tissue, including human endometrium, by the mere action of chopping and/or culture (33, 34). Even in the present study, it is possible that activation occurs during sectioning and incubation. Likewise, it is possible that TIMPs may be dissociated from the MMPs during processing.

MMPs can be activated by a number of proteases, including other MMPs. ProMMP-2 activation is thought to occur predominantly on the cell surface by the action of MT-MMPs (31) and such a mechanism is proposed here, given the cell surface location of some of the active gelatinase. ProMMP-9 can be activated by a number of proteases, including active forms of MMP-2 and MMP-13 (35). In endometrial explants in short-term culture, proMMP-9 is activated by MMP-3 (30) and in endometrial stromal cell cultures it can be activated by neutrophil elastase (12), whereas proMMP-3 can be activated by mast cell tryptase (11), which is released from activated mast cells perimenstrually (36). Endometrial mast cells themselves also produce MMP-1 (37). The very focal MMP activities shown here strongly support a role for leukocytes and their products in initiating MMP activation. Indeed, it is likely that, in vivo, a number of different activation mechanisms occur and that overall there is a cascade of MMP activation at menstruation.

Menstruation and ovulation (18) are among the rare events of tissue remodeling during normal adult life and in both of these MMPs are expressed and activated. However, in general, MMPs are associated with pathological conditions, such as tumor invasion, oral pathology and fibrotic diseases (reviewed in Ref. 25). It is important to note that the presence of active gelatinase in endometrial tumors, detected in our laboratory by the same technique of in situ zymography as that used here, is very much more widespread within the tumor tissue than is seen here at menstruation (38), suggesting that this normal process of tissue degradation is much more tightly regulated than is tumor invasion.

The data presented here moves us a further step in the transition from correlation to causality, in terms of defining a functional role for MMPs in the endometrium. By demonstrating that active MMPs are substantially increased in menstrual endometrium compared with other phases of the cycle and that the pattern of MMP activation is at discrete foci, as is the tissue breakdown, we have established that these MMPs are clearly in position to perform the matrix degradation characteristic of menstruation. However, it is still necessary to establish whether MMPs have a critical role in menstruation, by administration of specific inhibitors to menstruating women, either systemically or locally and by establishing whether such treatment modifies the timing, duration or magnitude of the menstrual process. MMP inhibitors are currently being administered systemically in humans in trials for late stage cancers and locally for treatment of periodontal disease (39). Given the high incidence of disorders of uterine bleeding in women, it is to be hoped that evaluation of such potential strategies for therapeutic intervention will be initiated in the near future.

Acknowledgments

We thank Sue Panckridge for assistance with the figures, Samantha Park for preparation of the final manuscript, and Dr. Rebecca Jones for critical review of the manuscript. We also thank Professor Gabor Kovacs and his patients for generously providing the endometrial tissue used in this study and Sr. Anne Clare and Heather Widjaja for its collection.

Footnotes

This work was supported by the National Health & Medical Research Council of Australia (Grants 143798 and 169003).

Abbreviations: MMP, Matrix metalloproteinase; TBS, Tris-buffered saline; TIMP, tissue inhibitors of metalloproteinases.

Received November 26, 2001.

Accepted February 5, 2002.

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