Regulated expression of matrix metalloproteinases, inflammatory mediators, and endometrial matrix remodeling by 17beta-estradiol in the immature rat uterus

All animal experiments were performed in compliance with NIH standards as specified in the Guide for Care and Use of Laboratory Animals (copyright 1996, National Academy of Sciences). Female Sprague-Dawley rats (Ace Animal Corp., Boyertown, PA) were ovariectomized at 21 days of age and allowed to recover for 3-5 days. For E₂ time course experiments, animals were administered a single physiological dose of E₂ (40 μg/kg in a 0.9% NaCl, 0.4% EtOH vehicle) by intraperitoneal (i.p.) injection at the indicated time intervals prior to tissue collection at necropsy. This in vivo dose level of E₂ has been shown to induce changes in uterine wet weight, tissue architecture, and gene expression characteristic of estrogen receptor activation [1, 46‐48]. For all other experiments, animals were i.p. administered a single 40 μg/kg bolus of E₂ 4 h prior to tissue harvest, while control animals received vehicle only in all studies. Batimastat was administered i.p. at a dose level (BB-9; 40 mg/kg in a 1× PBS, 0.1% Tween-20 vehicle) shown to be effective at inhibiting MMPs in vivo [49] 4 h prior to E₂ or saline control. Dexamethasone was administered i.p. at a dose level of 600 μg/kg in a 0.9% NaCl, 10% EtOH vehicle that has been previously used in rodent studies to assess in vivo steroid specific responses in the uterus [46‐48]. Animals were treated twice with dexamethasone (600 μg/kg) at 20 and 4 h before E₂ or saline control administration to elicit both early and late gene effects of the activated glucocorticoid receptor (GR) that may affect uterine responsiveness to E₂ via genomic or nongenomic mechanisms. Batimastat was generously provided by GlaxoSmithKline, while other treatment compounds were obtained from Sigma-Aldrich (USA). Dose levels of all compounds were administered at injection volumes of 1 ml/100 g body weight. Inhibitor compounds were administered i.p., with control animals receiving the corresponding vehicle solution i.p. at the same treatment times as inhibitor injections.

For uterine tissue collection at necropsy, animals were euthanized by decapitation on the same day in accordance with the specific treatment time interval and the tissue was stripped of fat and mesentery, weighed, and the horns separated. One horn from each animal was flash frozen in liquid N₂ and stored at -80°C for later RNA purification. For protein analyses, two isolated horns from different animals were pooled and fresh-processed for total protein extraction. For transmission electron microscopy analyses, tissues were immediately fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer.

Individual uteri were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, and 2 mm-cross sections of each uterine horn were cut and incubated overnight at 4°C in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Glutaraldehyde buffer was removed, and the samples were rinsed three times (15' each) in 0.1 M phosphate buffer. Samples were incubated in 1% osmium in 0.1 M phosphate buffer, rinsed, and dehydrated in a series of ethanol washes. Samples were incubated twice for 5' in propylene oxide and then transferred to a rotor for 1 h at room temperature in a 1:1 mixture of propylene oxide and epon [47% Embed 812, 31% DDSA (dodenyl succinic anhydride), 19% NMA (nadic methyl anhydride), 3% BDMA (benzyldimethylamine)], followed by an overnight incubation in 1:2 propylene oxide-epon, and finally 100% epon for 2-3 hrs. Individual uterine samples were embedded in 100% epon in silicon flat embedding molds, and capsules were polymerized in a 60°C oven for over 48 hrs. Ultrathin transverse sections (70 nm) were prepared using a diamond knife (Diatome) on a MT 6000-XL ultramicrotome, captured on 300-mesh copper grids, and stained with 2% uranyl acetate. All reagents and materials were obtained from Electron Microscopy Sciences (Hatfield, PA).

A minimum of 3 blocks with tissue from different areas along the length of each uterine horn were chosen at random from a total pool of 5-6 blocks and were used to prepare ultrathin sections to provide a comprehensive survey of whole organ ultrastructure representatively. A minimum of 6 transverse sections were prepared per tissue block and viewed using a Hitachi 7600 microscope fitted with an AMT digital camera. Visual analyses and micrograph surveys were restricted to the endometrial stromal compartment at a minimum of 3 random sites within the endometrium of each section so as to avoid bias. At each random site within the endometrial stromal matrix compartment, 3-6 digital photomicrographs at 12,000× magnification were taken. For quantitative analysis of collagen density, the full area of photomicrographs was digitally fitted with an 8 × 8 square grid containing a total of 64 cross-points, and each cross-point was evaluated to determine the structure that it overlaid. Image J software (NIH) was used to quantify the total number of crossbars landing on collagen fibrils (positive hits), and the values were expressed as a ratio to the total number of crossbars to determine a percent matrix density volume for each photomicrograph. Stromal areas with a high percentage of collagen fibrils cut in transverse were selected to best ensure that quantitative crossbar analysis was not significantly affected by dramatic differences in collagen fiber orientation. A total of 36-75 micrographs were analyzed per time point or treatment condition. Values for all micrographs for each animal were averaged, and statistical differences between averages of each time point or treatment group were analyzed using one-way ANOVA. Error bars on graphs represent standard error, and a standard P value of ≤ 0.05 was considered significant.

For TaqMan^® quantitative real-time PCR analyses of mRNA levels, total RNA was prepared individually from the uterus of each rat. Each uterus was disrupted for 2-3 minutes in 0.75 mL QIAzol lysis reagent (Qiagen, Valencia, CA) using a 5 mm bead in a TissueLyser (Qiagen-Retsch). The homogenate was subjected to a 0.2 mL chloroform extraction and centrifugation at 4°C for 15 min at 20,000 × g, and 0.35 mL aqueous phase was collected for RNA purification using an RNeasy kit (Qiagen) according to the manufacturer's protocol. Residual genomic DNA was removed by on-column RNase-free DNase treatment during RNA purification, and the RNA concentration was adjusted to 0.05 mg/mL. Messenger RNA levels were analyzed using quantitative real-time PCR on an ABI Prism 7900 Sequence Detection System and TaqMan^® gene expression assays (Applied Biosystems, Foster City, CA) for MMP-2 (Rn01538174_m1), MMP-3 (Rn00591740_m1), MMP-7 (Rn00563467_m1), MMP-9 (Rn00579162_m1), TNF-α (Rn01753871_m1), MCP-1 (Rn01456716_g1) and eukaryotic 18s rRNA (Hs99999901_s1). To compare mRNA expression levels among samples, mRNA for each gene of interest was normalized to the expression of a housekeeping gene, 18s rRNA, and differences in gene expression were calculated as a fold change to the mean of the vehicle group.

Detailed methods for protein extraction, SDS-PAGE, and immunoblotting have been described previously [46]. In brief, 2-3 uterine tissues were pooled and homogenized in 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 0.05% Brij 35, 1 mM PMSF, and 10 μg/ml leupeptin using a polytron homogenizer (Brinkman Instruments, Westbury, NY). Homogenates were centrifuged at 15,000 × g for 30 minutes at 4°C and the supernatants removed and protein quantified. Extracted total protein (20 μg) samples were loaded into the wells of a pre-cast gel (4-20% Tris-HCl; BioRad, Hercules, CA) and resolved by electrophoresis in a Mini-Protein II module (BioRad) according to the manufacturer's protocol. Resolved proteins were transferred to an Immobilon-P membrane (Millipore, Billerica, MA), which was blocked (5% BSA in 1× Tris-buffered saline + 0.1% Tween-20 + 2% normal goat serum) and then incubated in a 1:2000 dilution of affinity-purified polyclonal rabbit anti-MMP-2 IgG (Novus Biologicals, Littleton, CO; #NB200-193), anti-human MMP-7 IgG (Lifespan Bioscience, Seattle, WA; #LS-B1237), anti-MMP-3 IgG (Abcam, Cambridge, MA; #ab53015), anti-MMP-9 IgG (Abcam #ab7299), anti-TNF-α IgG (Cedarlane, Burlington, NC; #CL9572AP), anti-MCP-1 IgG (Millipore, Billerica, MA; #AB1834P) or monoclonal mouse anti-β actin IgG (Abcam #ab6276) overnight in blocking solution at 4°C with gentle agitation. Membranes were washed and incubated in a 1:10,000 dilution of secondary HRP-conjugated goat α-rabbit IgG or α-mouse IgG (Jackson Immunologicals, West Grove, PA) in blocking solution and then washed and transferred to a solution of SuperSignal Substrate reagents (Pierce/Thermo Scientific, Rockford, IL) for enhanced chemiluminescent signal detection, imaged, and analyzed using the FluorChem SA system (Alpha Innotech, San Leandro, CA) and AlphaEase FC software. Band intensities were measured as integrated density volumes (IDV) and expressed as % of control lane values for comparisons between treatment groups. Specificity of bands identified using primary antibodies was confirmed by exposing sample blots to either 2% normal goat serum alone, secondary goat HRP-conjugated antibody in the absence of primary antibody, or normal rabbit IgG (Jackson Immunologicals) only prior to secondary antibody incubation.

Extracted total protein (20 μg) samples were mixed 1:1 with zymography sample buffer (Bio-Rad) and incubated at room temperature for 10 min. Samples were loaded into the wells of a pre-cast gel (10% gelatin, BioRad) and resolved by electrophoresis in a Mini-Protein II module (BioRad) under non-reducing conditions. Active human MMP-2 (Calbiochem/EMD Biosciences, Gibbstown, NJ; #PF023) was loaded in one lane of the gel as a control. After electrophoretic separation, the gel was washed twice for 30 minutes each in 1× renaturation buffer (Bio-Rad) at room temperature (RT) with gentle agitation and then incubated in 1× development buffer (Bio-Rad) for 30 minutes at RT. Fresh development buffer was added and the gel incubated at 37°C for 18-24 hours. Developed gels were stained with Coomassie Brilliant Blue R-250 (0.5% in 30% methanol, 10% acetic acid) for 30 minutes at RT. The gel was destained in 30% methanol, 10% acetic acid until clear bands representing proteolytic areas were visualized. Gels were visualized with the FluorChem SA system (Alpha Innotech) and analyzed using AlphaEase FC software. Band intensities were measured as integrated density volumes (IDV) and expressed as % of control lane values for comparisons between treatment groups.

Data is represented as the average ± standard error of the mean (SE). Data were analyzed initially via one-way ANOVA followed by Tukey's Honestly Significant post-comparison test to assess differences between treatment groups. P values ≤ 0.05 were considered significantly different.

To better characterize previously described E₂-induced changes in uterine stromal collagen matrix remodeling [1], we developed a novel method to quantitatively assess changes in collagen fibril density using a grid overlay analysis of TEM photomicrographs (as described in Methods). This method establishes matrix density as a quantifiable endpoint for studies aimed at assessing changes in activity of matrix processing enzymes, including the MMPs, induced by bioactive compounds and inhibitors in a very precise and direct manner. We assessed collagen matrix density using micrographs prepared from uterine sections along the entire length of the uterine horn to best represent whole tissue architecture changes and over an extended time course after administration of a single physiological dose of E₂ (40 μ/kg) to study stromal collagen architecture throughout both the early hypertrophic and latter hyperplastic phases of uterine growth. Qualitative assessment of micrographs indicates that a dense irregular organization of collagen fibers is clearly observed within saline-treated tissues (0 h), but this organization rapidly disappears such that a significant difference is evident within 45 minutes of hormone administration. Matrix density reaches a low point by 4 h when only a few isolated fibrils within the stromal compartment remain (Figure 1). Quantitative analysis of collagen matrix density supports a significant decrease (~30% less than 0 h-saline levels) within 45 minutes post-E₂ treatment and a continual decline over time, reaching its lowest value (~70% less than 0 hr) at 4 h and remaining low through the 24 h treatment interval (Figure 2).

Upon establishing a quantitative method to assess changes in stromal matrix density, we assessed effects of a broad-range hydroxamate inhibitor of MMPs, batimastat (40 mg/kg), on E₂-induced matrix remodeling in OVX animals (Figure 3) to better characterize the role of activated MMPs in hormone-stimulated collagen reorganization. Matrix density was analyzed in saline- or batimastat-pretreated animals 4 h after E₂ administration, the time point at which collagen density was observed to be at its lowest after hormone treatment (Figures 1 and 2). Transmission electron microscopy assessment (Figure 3) shows that batimastat alone has no effect on the stromal collagen network (Sal-Sal vs. Bat-Sal), but overall collagen density in batimastat-pretreated E₂-induced animals (Bat-E₂) is significantly higher (~4-fold) compared to those receiving saline pretreatment and E₂ treatment (Sal-E₂), clearly indicating that E₂-induced uterine matrix remodeling is, at least in part, dependent upon activated MMPs.

While effects of ovarian hormones on uterine MMP expression have been studied under a variety of conditions in the rodent and human, we chose to fully characterize temporal changes in expression of MMP-2, MMP-3, MMP-7, and MMP-9 induced specifically by E₂ in the immature OVX rat and to examine any correlation in enzyme levels with the time course of collagen matrix remodeling. The enzymes surveyed include those known to be expressed by uterine epithelial (MMP-7) as well as stromal (MMP-2, MMP-3 and MMP-9) cells [50]. In addition, collagen substrates for MMP-2 (I, III, IV, V, VII, X, XI), MMP-3 (II, III, IV, V, VII, IX, X), MMP-7 (I, IV, X), and MMP-9 (IV, V, VII, XIV) have been characterized in vitro [50]. Levels of mRNA and protein for these MMPs were assessed following saline treatment (0 h) and at 5 intervals (4-72 h) post-E₂ administration. While mRNA transcripts for all MMPs analyzed (Figure 4A) are detected in the uterus in saline control tissues (0 h), significant increases in transcript levels are induced within 4 h by E₂ treatment for MMP-3, MMP-7, and MMP-9. Of these enzymes, the fold induction (~150-fold) for MMP-7 is clearly the most robust (Figure 4). In contrast, levels of MMP-2 mRNA do not increase until later in the time course, with peak fold induction (~7 fold) occurring at 48 h post-hormone administration, thereby placing it on a different time line of regulation compared to MMP-3, MMP-7, and MMP-9. Upregulation of mRNA for all enzymes is transient, as transcript levels decline significantly and return to control values by 72 h.

Western blot analyses (Figure 4B) were completed to determine if uterine MMP protein changes in correlation with the mRNA induction profiles for each enzyme. MMP-2, MMP-3, and MMP-9 are detected in saline tissues and increase after E₂ treatment in accord with peak changes in mRNA for each gene. Levels of the latent (~72 kDa) form of MMP-2 increase significantly within 4 h, but peak changes in latent and active (~64 kDa) protein do not occur until ~24-48 h post-E₂ treatment (Figure 4B), which correlates temporally with the peak in mRNA content (Figure 4A). Tissue levels of MMP-3 (~62 kDa) and MMP-9 (~90 kDa) increase within 4 h of E₂ treatment (Figure 4B) in conjunction with changes in mRNA (Figure 4A). While levels of MMP-9 decline over time, reaching control values within 24 h, MMP-3 protein remains elevated through 72 h, although the mRNA for this enzyme reaches control levels within 24 h after hormone administration (Figure 4A). This indicates that MMP-3 protein is temporally stabilized and levels do not decline to those observed prior to E₂ stimulation. Latent MMP-7 (~29 kDa) is not detectable in control tissues (0 h), but levels of the enzyme increase significantly by 4 h post-E₂ treatment, then decline over time to return to control values by 48 h (Figure 4B) in correlation to the temporal pattern of mRNA expression (Figure 4A). Upregulation of MMP-3, MMP-7, and MMP-9 protein expression 4 h post-E₂ indicates that these enzymes are in fact elevated rapidly by hormone treatment with peak increases in protein content occurring at the time point when stromal collagen matrix is lowest (Figures 1 and 2).

E₂-induced changes in activity of the gelatinase enzymes were assessed via gelatin zymography (Figure 5) to confirm overall protein expression levels observed via Western blot. The major bands representing gelatin-degrading activity were identified as active MMP-9 (~90 kDa) and latent and active MMP-2 (~72 kDa and 64 kDa, respectively). Overall activity of MMP-2 was greater than that of MMP-9 based upon band intensities. Levels of active MMP-2 decrease slightly within 4-12 h of hormone treatment (Figure 5). However, both latent and active forms of MMP-2 increase by 48 h post-E₂ treatment, with peak activity correlating temporally with the highest level of total protein observed via Western blot (Figure 4). Activity of MMP-9 increases significantly at 4 h post E₂-treatment (Figure 5) also corresponding to the time point when total protein levels are highest (Figure 4). Activity of MMP-3 and MMP-7 was assessed via casein gel zymography, however sensitivity of the assay was not sufficient to permit quantitative analysis (data not shown).

E₂-induced changes in expression of the pro-inflammatory cytokine, TNF-α, and the β-chemokine MCP-1, were assessed over the biphasic growth response to better characterize the tissue inflammatory response and to compare changes temporally with matrix processing and MMP expression. Levels of TNF-α mRNA and protein transiently increase with a peak in expression occurring 4 h after hormone treatment (Figure 6A). TNF-α mRNA increases nearly 25 fold at 4 h and remains elevated through 12 h post-E₂ treatment before declining to control values by 48 h. Western blot analysis (Figure 6B) identified the homotrimeric form of the protein (~51 kDa) as well as lower MW forms. Analysis of the receptor-ligand homotrimeric form shows slight elevation of protein early (1-2 h) with maximum levels occurring at 4 and 12 h post E₂ administration (Figure 6B), correlating temporally to the major time points of mRNA elevation. This pattern clearly indicates a potent overall enhancement of tissue levels of TNF-α expression, most notably during the early hypertrophic period of uterine growth following hormone treatment. MCP-1 mRNA expression is also significantly upregulated (~80 fold) 4 h post-E₂ treatment. While mRNA levels return to control values within 24 h, MCP-1 protein increases by 4 h but remains elevated through 48 h before returning to control tissue levels 72 h post-treatment (Figure 6B). Western blot analysis detects a higher MW form of MCP-1 (~20 kDa) which may represent an intact bioactive form of the chemokine, as well as a lower MW form (~16 kDa) which may represent a proteolytic and receptor-inactive product (Figure 6B). Blot analysis indicates that levels of the higher MW MCP-1 product peak at 4 h whereas the lower MW product peaks at ~24 h post treatment. E₂ treatment thus rapidly increases levels of two potent pro-inflammatory chemicals, TNF-α and MCP-1, and the induction correlates temporally with the hypertrophic phase of tissue growth, which is characterized by tissue edema and stromal matrix remodeling.

Because peak changes in matrix density and cytokine expression correlate temporally with the hypertrophic phase of E₂-induced uterine growth, it was of interest to assess the effects of the anti-inflammatory synthetic glucocorticoid, dexamethasone (600 μg/kg) on this inflammatory-like response. Dexamethasone pretreatment alone induces a decrease in uterine wet weight and significantly inhibits the typical increase in uterine wet weight effected by E₂ (Figure 7) in a manner consistent with previously published reports [34]. With respect to stromal matrix density, dexamethasone alone produces both a qualitative and quantitative change in matrix structure (Figure 8); there is a noticeable and statistically significant increase in stromal matrix density as compared to saline control animals (Dex-Sal vs. Sal-Sal). This indicates that dexamethasone modulates stromal matrix remodeling so as to generate a more densely organized collagen architecture in the immature uterus. In addition, dexamethasone pretreatment inhibits E₂-induced matrix remodeling; stromal collagen density is much higher (~4-fold) in dexamethasone-pretreated (Dex-E₂) animals as compared to saline-pretreated E₂-challenged animals (Sal-E₂). Quantitative analysis of collagen density indicates that collagen levels are equivalent in the Dex-E₂ as compared to control animals (Sal-Sal). It should be noted that the magnitude of inhibition of collagen matrix remodeling induced by E₂ is smaller in dexamethasone pretreated animals (Figure 8 bottom, Dex-E₂ vs. Sal-E₂) indicating that it is not fully efficacious at inhibiting the E₂ response.

In light of the inhibitory effect on E₂-induced stromal matrix remodeling (Figure 8), MMP mRNA and protein levels were analyzed in animals pre-treated with dexamethasone. Dexamethasone treatment increases expression of MMP-3 mRNA, when compared to levels in control animals (Figure 9A; Dex-Sal vs. Sal-Sal). In addition, dexamethasone does not inhibit the E₂-induced increase in MMP-3 mRNA (Figure 9A; Dex-E₂ vs. Sal-E₂). Levels of mRNA in Dex-E₂ animals are not significantly different from those in Dex-Sal controls, indicating that dexamethasone induction of mRNA expression for MMP-3 is not modified in the presence of E₂. Transcript levels for MMP-7 and MMP-9 are unaffected by pretreatment (Figure 9A, Dex-Sal vs. Sal-Sal), although dexamethasone pretreatment does significantly inhibit the E₂-induced upregulation of mRNA for these two enzymes (Dex-E₂ vs. Sal-E₂). Dexamethasone does not induce a change in MMP-2 mRNA (Figure 9A); however, effects on the E₂-induced upregulation of MMP-2 mRNA were not assessed since only the 4 h post-hormone treatment time point was studied, and transcript levels for this enzyme do not significantly increase until 48 h post-E₂ administration (Figure 4A).

Western blot analyses of MMP protein levels support the effects of dexamethasone on mRNA expression. Pretreatment inhibits E₂-induced upregulation of protein expression for MMP-3, MMP-7, and MMP-9 (Figure 9B; Dex-E₂ vs. Sal-E₂). Interestingly, levels of MMP-3 protein do not increase in response to dexamethasone pretreatment despite evidence of an elevation of tissue mRNA (Figure 9A). Gelatin zymography shows that dexamethasone pretreatment did not affect activity of MMP-2 or MMP-9 (Figure 10), although dexamethasone pretreatment does decrease the E₂-induced upregulation of MMP-9 total protein levels (Figure 9B). Because full activity of MMPs may not be recovered as a result of refolding of the enzymes after electrophoresis in gel zymography [51], it is unclear whether dexamethasone affects levels of active MMP-9 in vivo.

Dexamethasone potently inhibits the E₂-induced upregulation of MCP-1 mRNA and protein observed at 4 h post-hormone treatment (Figure 11). However, pretreatment does not affect E₂-induced upregulation of TNF-α mRNA (Figure 11A), though it does decrease protein content (Figure 11B).