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BOR - Papers in Press, published online ahead of print October 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020834
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BIOLOGY OF REPRODUCTION 70, 500–508 (2004)
DOI: 10.1095/biolreprod.103.020834
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Steroidal Regulation of Uterine Edema and Tissue Inhibitors of Metalloproteinase (TIMP)-3 Messenger RNA Expression Is Altered in TIMP-1-Deficient Mice1

Warren B. Nothnick2,3,4, Xuan Zhang3, and Han-E Zhou3

Departments of Obstetrics and Gynecology, Division of Basic and Clinical Women's Research,3 Molecular and Integrative Physiology,4 University of Kansas Medical Center, Kansas City, Kansas 66160


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue inhibitors of metalloproteinases (TIMPs) are expressed within the uteri of virtually all species where they are postulated to control extracellular matrix turnover, cellular apoptosis, and proliferation. The objective of the current study was to examine the steroidal regulation of uterine TIMP expression and to determine the potential role of the TIMP-1 gene product in this regulation. To accomplish these goals, ovariectomized female TIMP-1 wild-type and null mice were treated with estradiol, progesterone, or estradiol and progesterone and killed at various times after steroid administration. Estradiol induced a significant reduction in uterine TIMP-3 expression in wild-type mice at 8 and 24 h post-steroid administration, but the ability of this steroid to decrease TIMP-3 expression was impaired in the uteri of TIMP-1 null mice. Further, estrogen-induced uterine wet-weight gain/edema was enhanced in the TIMP-1 null mice, and the antiestrogen compound ICI 182,780 or progesterone could only partially block this estrogenic effect. It is concluded from this study that steroidal modulation of uterine TIMP-3 expression and regulation of wet-weight gain/edema are altered in TIMP-1 null mice. These observations suggest that steroids induce uterine TIMP-1 expression and, in turn, that TIMP-1 influences TIMP-3 mRNA expression and uterine edema.

estradiol, female reproductive tract, progesterone, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Within the uterus, the female sex steroids estrogen and progesterone play pivotal roles in the establishment of a suitable environment for embryo implantation and pregnancy. More specifically, these steroids regulate a multitude of cellular processes that include cell proliferation and differentiation as well as regulation of vascular permeability, angiogenesis, and adenogenesis [1–7]. To bring about these changes, estrogen and progesterone must appropriately modulate a variety of factors that include growth factors, cytokines, extracellular matrix proteins, and adhesion molecules [1–7]. One such family of factors that has been postulated to play a role in uterine physiology and the establishment of pregnancy is the tissue inhibitors of metalloproteinases, or TIMPs [8–16].

TIMPs are a multifunctional family of inhibitors of matrix metalloproteinases (MMPs) that include four distinct members: TIMP-1, TIMP-2, TIMP-3, and TIMP-4 [8, 17, 18]. All but TIMP-4 are expressed within the uterus of a variety of species that include mice [19], sheep [20], humans [21–25], and nonhuman primates [26, 27]. TIMPs are known modulators of cell proliferation, differentiation, and apoptosis [8, 17] and elicit these activities through processes that are either dependent on or independent of their ability to regulate MMP activity [17]. In the human, TIMPs are expressed within the uterus during the course of the menstrual cycle and have been postulated to play a role in the maintenance of endometrial integrity [21]. Studies using TIMP-1 null mice have suggested a similar role of this TIMP during the course of the rodent estrous cycle [19, 28], and loss of function of this TIMP is associated with subfertility [19, 28], reduction in reproductive life span [28], and altered uterine MMP [29] and TIMP [19] patterns of expression.

Uterine TIMPs expression appears to be regulated by steroids in a species-specific fashion. For example, in vivo studies in the cycling sheep uterus suggest that expression of TIMP-1 is down-regulated by estrogen, while that of TIMP-2 may be up-regulated by progesterone [20]. In contrast, in nonhuman primates, progesterone withdrawal [25, 26] is associated with a rapid increase in uterine TIMP-1 expression followed by a reduction in expression. Results from in vitro studies that incorporated human endometrial stromal cells are conflicting. Initial studies by Salamonsen and colleagues [27] suggested that withdrawal of progesterone did not influence TIMP-1 or TIMP-2 expression. A more recent study by this group [30] using a long-term culture of human endometrial stromal cells and progesterone analogs indicated that TIMP-1 expression was increased. A similar up-regulation of TIMP-3 by progesterone was found in vitro [23]. While the majority of the data would indicate that uterine TIMPs are regulated by steroids, data are conflicting. These deviations are most likely the result of differences in experimental designs (in vitro vs. in vivo studies, short-term culture vs. long-term culture) as well as possible species differences.

Our recent observation [19] that the pattern of murine uterine TIMPs fluctuate with the estrous cycle suggested to us, that like in other species, uterine TIMPs in the mouse might be regulated by steroids. This observation, coupled with the finding that uterine TIMP expression appears to be altered in TIMP-1 null mice [19], led to the following series of experiments that were designed to examine the steroidal regulation of uterine TIMPs expression and the contribution of TIMP-1 to this process. In the current study, wild-type and TIMP-1-deficient mice were utilized, and the role of estrogen and progesterone in regulation of uterine TIMPs expression was assessed. Unexpectedly, we found that although estrogen and progesterone regulated TIMP-1, TIMP-2, and TIMP-3 mRNA expression in wild-type uteri through what appeared to be classical steroid receptor-mediated mechanisms, regulation of TIMP-3 mRNA was altered in TIMP-1 null mice. Similarly, estrogenic regulation of uterine edema also appeared to be altered in the null mice. As such, these findings may be interpreted to suggest that steroidal induction of TIMP-1 within the uterus imparts a subsequent role for this TIMP in maintaining selective steroidal responses within this organ.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

TIMP-1 null and wild-type mice were utilized for all studies. TIMP-1-deficient animals (SVTER 129 background) were generated by homologous recombination of a neocontaining gene-targeting vector in mouse embryonic stem cells. Transmission of the mutant allele and the genotype of mice were determined by polymerase chain reaction analysis of the neosequences in genomic tail DNA. TIMP-1 deficiency was confirmed at the transcript and protein level by Northern analysis and protease inhibitor assays, respectively [19].

A breeding colony of both genotypes was generated at the University of Kansas Medical Center. Mice were housed within environmentally controlled conditions under the supervision of a licensed veterinarian. All animal procedures for these experiments were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee (IACUC). Mice were maintained on a 14L:10D cycle and provided water and mice chow ad libitum. Eight- to 12-wk-old female mice of both genotypes were ovariectomized and rested for 14 days, after which animals were randomly assigned to the respective treatment groups.

Administration of Treatments and Tissue Harvesting

Fourteen days after ovariectomy, wild-type and TIMP-1 null mice were injected s.c. with either vehicle (0.1 ml sesame oil), estradiol-17ß (E2, 10 µg/kg body weight [BW]), progesterone (P4, 100 mg/kg BW), or E2 + P4 (previous doses). Animals were then killed at 2, 4, 6, 8, and 24 h after steroid treatment by cervical dislocation, and uteri were removed, trimmed of fat and connection, and weighed. Uteri were then either snap frozen in liquid nitrogen or stored in RNAlater (Ambion Inc., Austin, TX) until utilized for RNA extraction.

To verify that E2 and P4 regulation of uterine TIMPs expression occurred via activation of their specific receptors, the E2 receptor antagonist ICI-182,780 (ICI; Tocris Cookson Inc., Ellisville, MO) and the P4 receptor antagonist RU-486 (Mifepristone; Dr. A.F. Parlow, NIDDK's National Hormone and Pituitary Program) were used. Mice were injected s.c. with either ICI (20 mg/kg dissolved in 100% ethanol and resuspended in sesame oil), RU-486 (20 mg/kg dissolved in 100% ethanol and resuspended in sesame oil), or vehicle (ethanol + oil). Thirty minutes later, mice received either oil vehicle, E2, P4, or E2 + P4 (previous doses), and mice were then killed at 6 (for TIMP-2), 8 (for TIMP-1), and 8 and 24 h (for TIMP-3) after steroid administration. These time points were chosen, as these were determined to be the times for maximal changes in uterine TIMP-1, TIMP-2, and TIMP-3 mRNA expression in the first experiment.

RNA Isolation

Total RNA was isolated from uteri by separately homogenizing the tissue in 1 ml of TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA) per 100 mg of tissue wet weight. RNA was then extracted with chloroform and precipitated with isopropyl alcohol according to the recommendations of the manufacturer. Total RNA samples were then electrophoresed through 1.0% agarose gels containing 2.2 M formaldehyde and were transferred to nylon membranes (Nytran, Schleicher and Schuell, Keene, NH) as recommended by the manufacturer. The murine TIMP-1, TIMP-2, TIMP-3, and TIMP-4 cDNA probes (kindly provided by Dr. Dylan Edwards, University of East Anglia, Norwich, UK) were excised from their respective plasmids with the appropriate restriction endonucleases, and the resulting inserts were labeled using Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Probes were labeled to a specific activity of 5 x 108 to 1 x 1010 dpm/µg of DNA using [{alpha}-32P] dCTP (Perkin-Elmer Life Sciences, Boston, MA). Filters were hybridized overnight, washed, and exposed to Blue Sensitive autoradiography film (Midwest Scientific, St. Louis, MO) for 24 h at -75°C. Hybridization signals were allowed to decay, and filters were subsequently hybridized for the 18S transcript using a rat cDNA probe (kindly provided by Dr. Michael Melner, Vanderbilt University, Nashville, TN) that cross hybridizes with the mouse transcript. In all experiments, TIMP data were normalized to the relative expression of the 18S transcript for each of the study groups. Data were digitized and quantitated using the GDS-8000 System (Ultra Violet Products, Upland, CA).

Statistical Analysis

All data were analyzed across treatment regimes within genotype by one-way ANOVA. When an F-test indicated statistical significance, post hoc analysis was done using the Student-Newman-Keuls procedure. Planned comparisons between genotypes within each specific treatment group were made using unpaired t-tests. Significance was set at P < 0.05 for all comparisons [31].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Steroidal Regulation of Uterine TIMP-1 Transcript Expression in Wild-Type Mice

TIMP-1 mRNA was not detected in the uteri of ovariectomized mice by Northern analysis using 10 µg of total RNA per lane. However, administration of exogenous E2 significantly increased TIMP-1 steady-state mRNA levels in a time-dependent manner. TIMP-1 transcript expression was detectable as early as 2 h post-E2 administration, peaked between 4 and 8 h post-E2 administration, and then decreased by 24 h after E2 administration (Fig. 1A). Administration of E2 plus P4 also induced a time-dependent increase in TIMP-1 steady-state mRNA expression, but this expression peaked earlier (6 h post-steroid administration) compared to E2 administration alone (Fig. 1B). Progesterone administered alone had no effect on uterine TIMP-1 expression, as levels remained undetectable by Northern analysis at all time points (data not shown). These data were interpreted to suggest that E2 but not P4 could increase uterine TIMP-1 steady-state mRNA levels. To verify that E2 action was mediated through its receptor proteins, a second experiment was conducted using the E2 receptor antagonist, ICI 182,780 (ICI). As expected, pretreatment of mice with ICI resulted in a significant reduction in uterine TIMP-1 steady-state mRNA expression in response to E2 treatment (Fig. 2). This suppression of TIMP-1 expression was also detected in mice pretreated with ICI followed by E2 + P4. In contrast, mice pretreated with the P4 antagonist RU-486 then challenged with E2 + P4 expressed similar levels of TIMP-1 transcript compared to E2 and E2 + P4 treated mice (Fig. 2). Finally, treatment with either ICI or RU-486 alone had no effect on uterine TIMP-1 transcription. Transcript expression was similar to vehicle-treated animals, as TIMP-1 mRNA steady-state levels were undetectable by Northern analysis (data not shown).



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  		FIG. 1. Steroidal regulation of uterine TIMP-1 mRNA expression in TIMP-1 wild-type mice. Mice were treated with either estrogen (A) or estrogen and progesterone (B) and killed at 0, 2, 4, 6, 8, or 24 h after steroid administration. Total mRNA was extracted and subsequently analyzed for TIMP-1 expression as described in Materials and Methods using 10 µg of total RNA/lane. TIMP-1 mRNA was detected as a single transcript of approximately 0.9 kb consistent with previous reports. TIMP-1 mRNA expression is expressed as the mean ratio of TIMP-1/18S transcript ± SEM and is reported as optical density (OD) units for four separate observations (n = 4 mice/treatment group). Autoradiographic exposures for TIMP-1 were for 24 h at -75°C, while that of 18S rRNA was for 1–3 h at -75°C. Different letters indicate statistical significance (P < 0.05) among treatment groups as determined by one-way ANOVA. A transcript consistent with that of TIMP-1 was not detected in the null mice (data not shown)



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  		FIG. 2. The effects of estrogen and progesterone receptor antagonists on uterine TIMP-1 expression. Animals were treated the estrogen receptor (ICI) or progesterone receptor (RU-486) antagonists followed 30 min later by treatment with either E2 or E2 + P4 (as indicated in the figure). Mice were killed at 8 h after steroid administration, and TIMP-1 mRNA expression was analyzed as described in Materials and Methods using 10 µg of total RNA/lane. TIMP-1 mRNA expression is expressed as the mean ratio of TIMP-1/18S transcript ± SEM and is reported as optical density (OD) units for five separate observations (n = 5 mice/treatment group). Autoradiographic exposures for TIMP-1 were for 24 h at -75°C. Different letters indicate statistical significance (P < 0.05) among treatment groups as determined by one-way ANOVA

Steroidal Regulation of Uterine TIMP-2 Transcript Expression in Wild-Type and TIMP-1 Null Mice

Both the 3.5- and the 1.0-kilobase (kb) transcripts for TIMP-2 were detected in the uteri of both wild-type and TIMP-1 null mice (Fig. 3, A and B). Levels of both transcripts were initially analyzed individually and did not significantly differ among treatment groups or between genotypes. As such, TIMP-2 expression is reported as the mRNA steady-state levels for both transcripts combined. In contrast to TIMP-1, TIMP-2 expression was highest in the uteri of wild-type ovariectomized (0 h, oil-treated) mice, and E2 administration decreased TIMP-2 expression. Specifically, E2 significantly reduced uterine TIMP-2 expression at 4, 6, and 8 h post-steroid administration compared to 0-h time points (Fig. 3A) with maximal suppression at 6 h after E2 treatment. By 24 h post-E2 administration, uterine TIMP-2 transcript expression returned to control levels (Fig. 3A). A similar pattern of TIMP-2 regulation was induced by E2 + P4 treatment, but the suppressive effect of both steroids was more evident at 4 h post-steroid administration compared to 6 h for E2 alone. In TIMP-1 null mice, the pattern of E2 and E2 + P4 regulation of uterine TIMP-2 mRNA expression was similar to that detected in wild-type mice with no significant differences in TIMP-2 transcript expression between genotypes within time points for either steroid treatments (Fig. 3, A and B). Finally, P4 alone did not influence TIMP-2 expression in mice of either genotype at any time point assessed (data not shown).



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  		FIG. 3. Steroidal regulation of uterine TIMP-2 mRNA expression in TIMP-1 wild-type and null mice. Mice were treated with either estrogen (A) or estrogen and progesterone (B) and killed at 0, 2, 4, 6, 8, or 24 h after steroid administration. Total mRNA was extracted and subsequently analyzed for TIMP-2 expression as described in Materials and Methods using 10 µg of total RNA/lane. TIMP-2 mRNA was detected as two transcripts of approximately 3.5 kb (upper band) and 1.0 kb (lower band) consistent with previous reports. TIMP-2 mRNA expression is expressed as the mean ratio of TIMP-2/18S transcript ± SEM for both transcripts combined (3.5 and 1.0 kb) and is reported as optical density (OD) units for four separate observations (n = 4 mice/genotype/treatment group). Autoradiographic exposures for TIMP-2 were for 24 h at -75°C. Different letters indicate statistical significance among the treatment groups as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice, while italics indicate comparisons within null mice). For all comparisons, P < 0.05 was considered statistically significant

To verify that steroidal modulation of uterine TIMP-2 steady-state mRNA expression was mediated through the respective steroid receptor proteins, mice were treated with ICI 182,780 and RU-486 and killed 6 h after steroid administration. Pretreatment with ICI 182,780 blocked the E2-induced down-regulation of TIMP-2 transcript expression in mice of both genotypes, as expression levels were similar to those of vehicle controls (Fig. 4). The E2 + P4 reduction in steady-state levels of TIMP-2 mRNA expression appeared to be due to E2 action but not P4. This suggestion is based on the observation that pretreatment with ICI blocked the E2 + P4 down-regulation of TIMP-2, but pretreatment with RU486 failed to restore TIMP-2 expression to those similar to the 0-h time point (Fig. 4). Finally, treatment with either ICI or RU-486 alone had no effect on uterine TIMP-2 steady-state mRNA levels (data not shown).



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  		FIG. 4. The effects of estrogen and progesterone receptor antagonists on uterine TIMP-2 expression. Animals were treated the estrogen receptor (ICI) or progesterone receptor (RU-486) antagonists followed 30 min later by treatment with either E2 or E2 + P4 (as indicated in the figure). Mice were killed at 6 h after steroid administration, and TIMP-2 mRNA expression was analyzed as described in Materials and Methods using 10 µg of total RNA/lane. TIMP-2 mRNA expression is expressed as the mean ratio of TIMP-2/18S transcript ± SEM and is reported as optical density (OD) units for four separate observations (n = 4 mice/treatment group). Autoradiographic exposures for TIMP-2 were for 24 h at -75°C. Different letters indicate statistical significance (P < 0.05) among treatment groups as determined by one-way ANOVA

Steroidal Regulation of Uterine TIMP-3 Transcript Expression in Wild-Type and TIMP-1 Null Mice

TIMP-3 was detected as a major transcript of 4.5 kb in the uteri of both wild-type and TIMP-1 null mice (Fig. 5). In wild-type mice, E2 administration induced a slight but significant increase in uterine TIMP-3 expression at 2, 4, and 6 h after steroid treatment. At 8 and 24 h, E2 significantly reduced uterine TIMP-3 steady-state mRNA levels to at or below the 0-h levels (Fig. 5A). In TIMP-1 null mice, E2 again induced increases at 2, 4, and 6 h post-steroid administration. However, in contrast to TIMP-3 expression in wild-type mice, E2 treatment did not suppress uterine TIMP-3 steady-state mRNA levels at 8 h post-steroid administration, but a reduction was noted by 24 h post-E2 treatment (Fig. 5). Administration of E2 + P4 resulted in a similar pattern of TIMP-3 expression with increases in transcript steady-state levels at 2, 4, and 6 h after administration. Again at 8 h posttreatment, TIMP-3 expression was not suppressed by E2 + P4 in the null mice as it was in the wild-type mice. By 24 h post-steroid administration, TIMP-3 expression was reduced compared to the 2-, 4-, or 6-h groups in mice of both genotypes. Further, the reduction of TIMP-3 expression by E2 + P4 was not as marked as it was for E2 alone in mice of either genotype, indicating that at 24 h, P4 may be capable of blocking E2 down-regulation of TIMP-3 steady-state mRNA levels. Finally, P4 alone did not influence TIMP-3 steady-state mRNA levels in mice of either genotype at any time point assessed (data not shown).



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  		FIG. 5. Steroidal regulation of uterine TIMP-3 mRNA expression in TIMP-1 wild-type and null mice. Mice were treated with either estrogen (A) or estrogen and progesterone (B) and killed at 0, 2, 4, 6, 8, or 24 h after steroid administration. Total mRNA was extracted and subsequently analyzed for TIMP-3 expression as described in Materials and Methods using 10 µg of total RNA/lane. TIMP-3 mRNA was detected as a major transcript of approximately 4.5 kb consistent with previous reports. TIMP-3 mRNA expression is expressed as the mean ratio of TIMP-3/18S transcript ± SEM and is reported as optical density (OD) units for six separate observations (n = 6 mice/genotype/treatment group). Autoradiographic exposures for TIMP-3 were for 8 to 12 h at -75°C. Different letters indicate statistical significance among the treatment groups as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice, while italics indicate comparisons within null mice). Asterisks (*) indicate statistically significant differences between genotypes within treatment group by planned comparisons using unpaired t-tests. For all comparisons, P < 0.05 was considered statistically significant

To verify that steroidal modulation of uterine TIMP-3 was mediated through the respective steroid receptor proteins, mice of both genotypes were treated with ICI 182,780 or RU-486, challenged with steroids, and killed 8 and 24 h following steroid administration. In wild-type mice, E2 induced an approximate 35% decrease (P > 0.05) in TIMP-3 mRNA expression at 8 h posttreatment (Fig. 6A), which was in agreement with time course studies (Fig. 5). Pretreatment with ICI 182,780 blocked this E2-induced decrease in wild-type mice. In contrast, E2 did not influence TIMP-3 expression in null mice at 8 h posttreatment, as transcript levels were similar to vehicle treated mice (Fig. 6A). When compared within treatment between genotypes, E2 induced a significant reduction in TIMP-3 mRNA expression in the wild-type mice (Fig. 6A; compare +/+ to -/- E2 groups). Finally, treatment with E2 + P4 alone or with antagonists did not influence TIMP-3 mRNA steady-state levels in mice of either genotype (Fig. 6A).



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  		FIG. 6. The effects of estrogen and progesterone receptor antagonists on uterine TIMP-3 expression. Animals were treated the estrogen receptor (ICI) or progesterone receptor (RU-486) antagonists followed 30 min later by treatment with either E2 or E2 + P4 (as indicated in the figure). Mice were killed at 8 h (A) and 24 h (B) after steroid administration, and TIMP-3 mRNA expression was analyzed as described in Materials and Methods using 10 µg of total RNA/lane. TIMP-3 mRNA expression is expressed as the mean ratio of TIMP-3/18S transcript ± SEM and is reported as optical density (OD) units for five separate observations (n = 5 mice/treatment group). Autoradiographic exposures for TIMP-3 were for 8 h at -75°C. Different letters indicate statistical significance among the treatment groups as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice, while italics indicate comparisons within null mice). For all comparisons, P < 0.05 was considered statistically significant

At 24 h, E2 induced a significant reduction in TIMP-3 steady-state mRNA levels in mice of both genotypes, and this level of expression did not differ between genotypes (Fig. 5). Pretreatment with ICI confirmed that this reduction was via an E2 receptor-specific pathway in both wild-type and null mice (Fig. 6B). Also in accord with the time course studies (Fig. 5), E2 + P4 treatment restored TIMP-3 steady-state mRNA levels similar to those of vehicle-treated mice and greater than those levels detected in mice treated with E2 alone (Fig. 6B). This regulation was similar in mice of both genotypes and suggests that P4 could block the E2-induced suppression. This was confirmed by RU-486 pretreatment, as TIMP-3 steady-state mRNA levels were significantly reduced in the E2 + P4 + RU486 group compared to the E2 + P4 group in both wild-type and null mice (Fig. 6B).

Steroidal Regulation of Uterine TIMP-4 Transcript Expression in Wild-Type and TIMP-1 Null Mice

TIMP-4 was not expressed within the uterus of either wild-type or TIMP-1 null mice under control or any hormonal treatment regime (data not shown). In accord with our previous findings [32], positive ovarian control tissue did express TIMP-4 mRNA (data not shown), verifying in fact that uterine TIMP-4 transcript is undetectable by Northern analysis using 10 µg of total RNA.

Estrogen Induction of Uterine Wet-Weight Gain Is Partially Independent of Estrogen Receptor-Specific Pathways in TIMP-1 Null Mice

As reproductively cycling TIMP-1 null mice have larger uteri compared to wild-type counterparts [19], the effect of steroid treatment on uterine wet-weight gain/edema was assessed. E2 significantly increased uterine wet weight above vehicle levels in mice of both genotypes (Fig. 7). Comparison between genotypes within treatment group revealed that this E2-induced increase in wet weight was significantly greater in the TIMP-1 null mice compared to wild-type counterparts. In addition, the antiestrogen ICI 182,780 blocked the E2-induced increase in uterine wet weight in wild-type mice, reducing uterine weight wet to similar values compared to vehicle-treated mice. In the null mice, ICI reduced uterine wet weight, but these weights were still significantly greater compared to vehicle-treated null mice (Fig. 7). Comparison between genotypes within treatment group revealed that ICI could only partially block the E2 induction of uterine wet-weight gain.



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  		FIG. 7. Effects of steroids and steroid receptor antagonists on uterine wet weights between genotypes. Mice were killed 8 h after treatment administration, and uterine wet weights were determined. Uterine wet weights are expressed as percentage of body weight and are expressed as the mean ± SEM for six mice/genotype/treatment group (n = 6). Different letters indicate statistical significance among the treatment groups as determined by one-way ANOVA (block letters indicate comparisons within wild-type mice, while italics indicate comparisons within null mice). Asterisks (*) indicate statistically significant differences between genotypes within treatment group by planned comparisons using unpaired t-tests (* = P < 0.05, ** = P < 0.01, *** = P < 0.001). For all comparisons, P < 0.05 was considered statistically significant

Administration of E2 + P4 reduced uterine wet weight compared to E2 treatment alone in wild-type mice (Fig. 7). However, compared to E2 treatment alone in the null mice, the combination of these steroids did not significantly reduce uterine wet weights (Fig. 7). Comparison between genotypes within treatment group further revealed that E2 + P4 regulation of uterine wet weight was altered, as null mice had significantly greater uterine wet weights (Fig. 7). Administration of ICI in combination with E2 + P4 reduced uterine wet weight compared to E2 + P4 in mice of both genotypes, but again there was a significant alteration in regulation of uterine wet weights between genotypes. Finally, administration of the P4 receptor antagonist RU486 (Mifepristone) in combination with E2 + P4 resulted in an increase in uterine wet weight in both wild-type and TIMP-1 null mice, and this increase was significantly greater in the null mice (Fig. 7). Overall, it appears that E2, in the face of either P4 or the E2 receptor antagonist ICI, has a more profound effect on uterine wet weight in the TIMP-1 null mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The pattern of expression of uterine TIMPs during the menstrual [21, 22, 24–26] and estrous [19] cycles suggests that these factors might be under the transcriptional regulation of E2 and or P4. In vivo and in vitro studies suggest that this is true, but there appears to be species-specific differences in the effects of these steroids on TIMP expression [20–28]. In the current study, we support our previous postulation [19] that, in the mouse, uterine TIMP expression is regulated by the steroids E2 and P4. In addition to providing further evidence for steroidal regulation of uterine TIMPs, this study makes several novel and important observations that include distinct patterns of steroidal regulation for TIMP-1, TIMP-2, and TIMP-3 and a potential role for TIMP-1 in the modulation of subsequent steroidal effects within the uterus.

In the current study, it was demonstrated that uterine expression of TIMP-1 is up-regulated by E2 alone or in combination with P4. The ability of the estrogen receptor antagonist ICI 182,780 but not the progesterone receptor antagonist RU-486 to block E2 (or E2 + P4) induction of TIMP-1 confirms the specificity of the E2 induction of this TIMP. The precise role of TIMP-1 within the uterus of nonmenstruating species is largely unknown, but disruption of the gene is associated with reproductive abnormalities [19, 28]. Determination of the temporal pattern of uterine TIMP-1 expression may provide insight into the role of this TIMP within the uterus. In the current study, the temporal pattern of E2-induced uterine TIMP-1 expression parallels that of the immediate early responses induced by E2, such as water imbibition/edema and macromolecular uptake [33, 34]. When TIMP-1 null mice were treated with E2, the increase in uterine edema (assessed at 8 h post-E2 administration as uterine wet weight) was approximately 50% greater compared to wild-type counterparts, suggesting that TIMP-1 may functionally control the extent of uterine edema.

The mechanisms by which TIMP-1 may regulate uterine edema may rely on the ability of this TIMP to regulate MMP activity. Compared to wild-type mice, uterine MMP activity is elevated in TIMP-1 null mice [29; unpublished results], and this elevation in active MMP may play a role in the induction of uterine edema. Recent evidence suggests that estrogen induction of uterine edema is mediated at least in part through the action of vascular endothelial growth factors (VEGF) [35, 36]. Further evidence indicates that VEGF up-regulates MMP expression in vascular smooth muscle [37] and endothelial cells [38], and it is well established that MMPs themselves are potent inducers of vascular permeability [39, 40]. As such, in the current study, the absence of TIMP-1 may lead to elevated MMP activity (compared to wild-type mice), which in turn leads to enhanced uterine edema via the action of these MMPs.

One of the more interesting findings of the current study was the observation concerning the regulation of uterine edema. More specifically, E2-induced uterine edema could be blocked in the wild-type mice by either ICI or P4, but this inhibition was only partially blocked in the null mice. This observation may suggest that disruption of the TIMP-1 gene product alters the mechanism by which E2 regulates uterine edema and shifts the mechanism from an estrogen receptor-dependent mechanism (blocked by ICI or P4) to that of an estrogen receptor-independent mechanism (not or only partially blocked by ICI or P4). It is well established that E2 actions are mediated through the nuclear estrogen receptors ER-{alpha} and/or ER-ß [41] and that ICI and P4 can block E2 action via inhibition of signaling through E2 receptors [42, 43]. However, recent accumulating evidence suggests that steroid hormones can elicit nongenomic actions within reproductive tissues [44]. Within the uterus, E2 has been shown to stimulate calcium entry into endometrial cells [45] and tissue [46, 47], and this mechanism is thought to play a role in normal uterine physiology. Pertinent to the current study is the observation that an increase in intracellular calcium has been associated with increases in MMP activity [48–50] and that MMPs themselves [51] can further increase calcium influx into cells. Other potential mechanisms that may stimulate the transcriptional activity E2 receptors independent of E2 itself may include cross talk between growth factors and the E2 receptor [52]. Elevated uterine MMP activity, which is characteristic of the TIMP-1 null mice [29], may lead to the liberation of free biologically active growth factors that could stimulate E2 receptor-dependent pathways independent of the presence of this steroid. We propose that in the TIMP-1 null mice, elevated MMP activity may lead to an enhanced action of calcium or growth factors (whose biological activity can be increased by MMP actions [53, 54]) that in turn may regulate E2 induction of uterine edema independent of the classical nuclear E2-dependent E2 receptor pathway.

Another interesting observation was the regulation of uterine TIMP-2 and TIMP-3 expression between the wild-type and TIMP-1 knockout mice. In mice of both genotypes, TIMP-2 expression was high in 0-h control mice; decreased to the lowest levels in response to either E2 or E2 + P4 at 4 and 6 h post-steroid administration, respectively; and then returned to baseline (0 h) levels at 24 h. Furthermore, this steroidal regulation could be blocked by the E2 receptor antagonist ICI in mice of both genotypes. Collectively, these data suggest that steroidal regulation of uterine TIMP-2 is similar in wild-type and null mice and that the presence or absence of TIMP-1 does not influence the expression of TIMP-2. E2 appears to down-regulate TIMP-2 expression, while P4 has no effect on its expression. While we interpret these data to suggest that E2 decreases steady-state levels of TIMP-2 mRNA, we are aware that there could actually be an increase in TIMP-2 translation and protein expression. This would suggest that the end result of E2 treatment would be an increase in TIMP-2 activity.

In contrast to TIMP-2 regulation, steroidal regulation of uterine TIMP-3 expression appears to differ between wild-type and TIMP-1 null mice, but only after 8 h post-steroid administration were these differences in regulation between genotypes noted. In wild-type mice, E2 induced a decrease in TIMP-3 expression at 8 and 24 h post-steroid administration, but in the null mice a reduction in TIMP-3 expression did not occur until 24 h post-E2 administration. One interpretation of this observation may be that the regulatory mechanism by which uterine TIMP-3 expression is modulated is out of phase or delayed in the TIMP-1 null mice. This postulate is supported by the observation in the current study that at 8 h post-E2 treatment, TIMP-3 mRNA steady-state levels begin to decrease in the wild-type mice, and this decrease can be blocked by pretreatment with the E2 receptor antagonist ICI. In contrast, E2 does not suppress TIMP-3 expression at 8 h in the null mice, and ICI pretreatment has no effect on steady-state mRNA levels of this TIMP. However, by 24 h post-E2 treatment, TIMP-3 mRNA steady-state levels are significantly reduced in mice of both genotypes, and this reduction can be blocked by ICI. While the mechanism(s) for this "delayed" regulation of TIMP-3 expression is unknown, alterations in calcium or growth factor signaling that result from the absence of TIMP-1 may be responsible.

In summary, uterine TIMP-1 is regulated by both E2 and E2 + P4. It appears that while disruption of the TIMP-1 gene is not associated with an alteration in steroidal regulation of uterine TIMP-2 expression, it is associated with an altered regulation of uterine TIMP-3 expression. Estrogen up-regulation of TIMP-1 and down-regulation of TIMP-2 (and to a lesser extent that of TIMP-3) occur concurrent to the induction of uterine wet-weight gain/edema, indicating that these TIMPs may play a role in regulating this process. ICI blocks estrogen regulation of TIMP-1, TIMP-2 expression, and uterine edema, indicating a similar estrogen pathway via classical ER. Precise roles of TIMP-1 and TIMP-2 in regulating uterine edema are uncertain but may depend on their MMP-dependent or MMP-independent actions. Based on data with the TIMP-1 null mice, we postulate that TIMP-1 controls the extent of edema, as absence of this inhibitor was associated with a more pronounced increase in uterine edema. The fact that this edema was only partially blocked by ICI or P4 in the null mice suggests a complicated role for TIMP-1 that occurs in response to estrogen treatment. Possibilities include elevation of MMP activity, which in turn could alter bioavailability/bioactivity of growth factors/cytokines, which could influence edema.


   FOOTNOTES
 
1 This research was supported by NIH grant award HD39765 to W.B.N. Back

2 Correspondence: Warren B. Nothnick, University of Kansas Medical Center, Department of Obstetrics and Gynecology, 3901 Rainbow Blvd., Kansas City, KS 66160. FAX: 913 588 6271; wnothnic{at}kumc.edu Back

Received: 9 July 2003.

First decision: 25 July 2003.

Accepted: 14 October 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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