1999 Progress Report: Dioxin and Steroid Regulation in an Endometriosis Model

EPA Grant Number: R826300
Title: Dioxin and Steroid Regulation in an Endometriosis Model
Investigators: Osteen, Kevin G.
Institution: Vanderbilt University School of Medicine
EPA Project Officer: Aja, Hayley
Project Period: December 15, 1997 through December 14, 2000
Project Period Covered by this Report: December 15, 1998 through December 14, 1999
Project Amount: $381,404
RFA: Endocrine Disruptors (1997) RFA Text |  Recipients Lists
Research Category: Environmental Justice , Endocrine Disruptors , Human Health , Safer Chemicals


Endometriosis is a persistent, steroid-mediated disease which develops in 10-15 percent of women in industrialized nations, most often from the ectopic implantation of endometrial fragments that enter the peritoneum by retrograde menstruation. The environmental contaminant 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD or dioxin) has been shown by numerous laboratories to disrupt steroid action in reproductive tissues. This environmental agent also has been linked to the development of endometriosis in primates; however, the biological mechanism(s) that account for TCDD's association with this disease is not known. The long-term objectives of our research grant is to identify which specific cellular and molecular mechanisms link TCDD disruption of steroid action to endometriosis. Our preliminary data suggest that, as a consequence of TCDD exposure, progesterone-mediated regulation of matrix metalloproteinase (MMP) expression may be disrupted in the human endometrium. Endometrial MMPs are necessary for mediating the extensive tissue turnover that is a component of the normal menstrual cycle. Unfortunately, these enzymes also may provide the necessary matrix breakdown that allows endometrial fragments to penetrate the peritoneal wall during establishment of endometriosis. Our laboratory has previously demonstrated that blocking the expression or action of these enzymes can prevent the ectopic implantation of human endometrial tissue in an experimental model of endometriosis in nude mice.

Our grant seeks to determine if TCDD can interfere with steroid-mediated MMP regulation and whether this interference plays a role in the establishment of experimental endometriosis. The project is divided into three specific objectives. The first objective will determine the expression pattern of several cell-specific MMPs in endometriotic lesions from women versus lesions removed from our nude mouse model. These studies will identify any alterations in specific MMP expression that might occur following treatments of human tissue or nude mice with TCDD. The second objective is designed to investigate the cell-specific mechanisms by which TCDD can disrupt progesterone-mediated suppression of MMPs. Blocking the action of progesterone would prevent the important role progesterone normally plays in suppressing these enzymes during the menstrual cycle. The third objective will investigate the role of TCDD in altering cell-type specific response of endometrial cells to local immune cytokines, which can stimulate endometrial MMP expression in the inflammatory-like environment within the peritoneal cavity

Progress Summary:

The three objectives of this research project are designed to be interactive and, therefore, elements of each have been carried out during the first 2 years of the grant period. For this reason, our report will detail specific focus areas of research as opposed to being divide strictly according to stated yearly objectives.

Analysis of In Vivo MMP Expression Patterns. We have focused considerable efforts into acquiring tissue and lesions from women with endometriosis for in situ hybridization studies, and this process continued in the second year. In contrast to normal endometrial tissue, we have found MMP-3, MMP-7, and MMP-11 mRNA to be variably expressed in endometriotic lesions with no apparent cyclicity. Moreover, MMP-11 mRNA is very highly expressed in both eutopic and ectopic tissues from women with endometriosis. Increased expression of MMP-11 mRNA in "apparently normal" endometrium from women with endometriosis compared to women without the disease, suggests this MMP may play a role in the development of endometriosis. Lack of cyclic variability of MMP expression in endometriosis also has been demonstrated in vitro. Tissues removed during the proliferative phase of the cycle were maintained 48 hours in organ culture with estradiol or estradiol plus progesterone. Although in vitro progesterone treatments normally suppress mRNA for MMP-3, MMP-7, and MMP-11, expression of all three mRNAs was significantly higher in endometriosis cultures regardless of steroid treatment.

Normally, stromal cell MMP-3 and MMP-11 expression is mediated by progesterone, whereas epithelial MMP-7 expression is paracrine mediated (via TGF- ). In the first year of our studies, we reported that the misexpression of MMP-3 and MMP-7 in endometriosis appears to be correlated with increased local expression of interleukin-1 (Publication 1). The expression of mRNA for MMP-3 at ectopic sites is variable and appears to exhibit less extensive expression than mRNA for MMP-7. These observations can now be extended to include MMP-11 mRNA and, taken together, suggest that local paracrine factors may play an important role in promoting broad MMP misexpression or overexpression at ectopic sites of endometrial growth.

Through the remainder of the grant, we will continue collecting endometrial and endometriotic samples from surgeries performed in the Women's Reproductive Health Clinic at Vanderbilt. Expression of MMPs and regulatory cytokines are being assessed in human endometriosis tissues for comparison with expression in human tissue removed from experimental endometriosis in nude mice. Additionally, we have recently acquired primate samples from the Endometriosis Association and will quickly begin to further examine expression patterns of MMPs and cytokines in these tissues. Because we have available endometriosis tissues from both TCDD-exposed and non-exposed animals, we will attempt to define abnormal patterns of cytokine expression associated with TCDD exposure. If a cytokine(s) or growth factor(s) is identified that appears to correlate with TCDD exposure, we will then use the nude mouse model to attempt to determine whether or not these factors play an important role in the misexpression of MMPs we have correlated to the development of experimental endometriosis.

Cellular Mechanisms of TCDD Action. Preliminary observations indicate that exposing human endometrial organ cultures to TCDD can prevent progesterone suppression of MMP-3 or MMP-7. Because progesterone-mediated suppression of MMPs prevents development of endometriosis in nude mice, we need to identify the specific cellular mechanism by which TCDD prevents progesterone suppression of MMPs. We know that progesterone regulates TGF- 2 expression and action in the endometrium that is critical for suppression of MMP-7, and that blocking this growth factor also will impact patterns of MMP-3 expression. During the first year of the grant, using our nude mouse model, we were able to demonstrate that antibody treatments that block TGF- action greatly reduced the ability of progesterone treatments to prevent experimental endometriosis (Publication 2). Importantly, we demonstrated that in vitro TCDD exposure prevented the increase in endometrial TGF- 2 protein expression that normally follows exposure to progesterone (Publication 3). In year two studies, we have further demonstrated that TCDD inhibition of TGF- 2 occurs at the level of the gene because progesterone associated increases in TGF- 2 mRNA can be blocked by TCDD (manuscript in preparation). We believe that the ability of TCDD to prevent progesterone induction of TGF- 2 may be a critical component of this toxin's ability to alter the normal pathway by which progesterone regulates MMP expression. In the second year of the grant, we began to explore in more depth the precise molecular mechanism by which progesterone may regulate TGF- 2 expression in the absence of a known progesterone response element. Our studies to date, using Hela cells transfected with the B form of the progesterone receptor (PR B) and a 3.1 Kb piece of the TGF- 2 promoter fused to a chloramphenicol acetyl transferase (CAT) reporter construct, indicate a possible interaction between the progesterone receptor and the TGF- 2 promoter (Publication 4). Efforts in the coming year will continue to investigate this possible interaction so that the effect of TCDD on this system can be determined.

A number of investigators have demonstrated an important role for TGF- 2 in increasing expression of TIMP-1 protein. TIMP-1, a natural inhibitor of MMP action, is increased in the endometrium during the secretory phase. Using western protein analysis of endometrial organ cultures, we have demonstrated increased TIMP-1 expression following treatment with either TGF- 2 or progesterone. Cultures simultaneously treated with TCDD do not secrete increased levels of TIMP-1, indicating that this toxin not only blocks steroid-mediated suppression of MMPs but also can prevent increases in TIMP-1, an important, locally produced natural inhibitor of MMP action. Importantly, we have demonstrated that the above effects of TCDD can be observed at in vitro doses as low as 10-12 M.

Additionally, we previously demonstrated that retinoic acid (RA) is synthesized locally in the normal human endometrium and that this synthesis can be regulated by progesterone during the secretory differentiation of human endometrial stromal cells (Publication 4). We were excited to find that RA, in the absence of progesterone treatment, can both suppress endometrial MMP expression and prevent development of experimental endometriosis (Publication 1). In the second year of the grant, we have found that RA, like progesterone, can induce TGF- 2 protein and mRNA, and that this expression is critical to suppression of MMPs by RA. Moreover, as observed following progesterone exposure, TCDD can block RA-associated increase in the expression of TGF- 2 protein and mRNA. Although we have not yet determine the ability of

TCDD to effect RA prevention of experimental endometriosis, these preliminary studies strongly indicate that this toxin also will disrupt this effect of RA.

Nude Mice Studies. To date, we have demonstrated that 90 percent of mice injected with human tissues that were treated in vitro with TCDD develop ectopic lesions regardless of whether or not endometrial tissues also were treated with progesterone. Lesions removed from mice receiving TCDD-treated tissue also appear to be larger and more numerous than lesions found in mice receiving tissue that was not exposed to TCDD. These observations are similar to findings reported by others (Rier, et al. Fund App Tox 1993;21:433; Cummings, et al. Fund App Pharm 1996;138:131) that describe a more aggressive disease following exposure to TCDD. Further studies are exploring the mechanisms that may be responsible for the apparent increase in severity of disease associated with TCDD.

We need to better understand the mechanism of the impact that TCDD exposure has on the development of experimental endometriosis. As noted above, in situ hybridization studies are being performed to compare expression of MMP-3 and MMP-7 mRNA in endometrial lesions removed from women with the disease versus the expression of these enzymes in human tissue removed from nude mice (to date, studies include control animals versus animals receiving TCDD-treated human tissues). In addition to the differences in MMP-3 versus MMP-7 mRNA localization, differences in ectopic lesion expression of the inflammatory cytokine IL-1 are apparent. We find that TCDD-mediated disruption in MMP expression in human endometrial organ cultures is accompanied by an increase in the in vitro expression of IL-1 . In vivo, we find that tissue treated in vitro with TCDD and subsequently injected into nude mice continue to demonstrate increased staining for IL-1 , when compared to tissues that were not exposed to TCDD in vitro. Endometriotic lesions removed from women with the disease showed a variable intensity of staining for IL-1 when compared to normal endometrial tissue removed at the same time. Nevertheless, because a number of laboratories, including ours, has shown that IL-1 exposure can interfere with progesterone-mediated endometrial cell differentiation, our early results indicate that local IL-1 expression may play a role in endometriosis.

Additional Comments. Over the last year, we have made significant progress in overcoming an important difficulty with the nude mouse studies. Lesions removed from mice are very small; the largest of lesions being only 3-4 mm. Extraction of total RNA typically yields 0.5-3.0 mcg, depending on lesion size. Northern analysis is not feasible and although in situ hybridization studies are possible, these can be performed using only a limited number of probes. We have now designed human-specific primers for use in semi-quantitative RT-PCR, which have enabled us to screen very small amounts of RNA for MMP-3, MMP-7, MMP-11, TGF- 2, and IL-1 . Expression of these RNAs are normalized to human cytochrome oxidase-1 (co-1), a mitochondrial enzyme that is not cyclically regulated in the endometrium. This technique should greatly enhance our ability to target specific factors that are responsive to TCDD and associated with the development of endometriosis. Once these factors are identified, in situ hybridization can be performed to localize expression of a targeted gene to a particular cell type or area of the lesions (i.e., peri-glandular).

Future Activities:

We will continue to explore the role of TCDD disruption of progesterone action. We are particularly interested in the mechanisms by which TCDD prevents progesterone induction of TGF- 2. A number of investigators have demonstrated the importance of an "E-box" located within the TGF- 2 promoter, which is essential for induction of this gene. Previous studies also have shown the TGF- 2 E-box to be highly homologous to the xenobiotic response element (XRE). The activated TCDD receptor complex (AhR/ARNT) binds to the XRE within responsive genes to mediate its effects. We have begun studies to determine whether the progesterone receptor also can interact with the E-box/XRE (either directly or indirectly through other transcription factors) to regulate TGF- 2 expression. If this hypothesis proves correct, it would be possible that TCDD could block the effect of progesterone simply by occupying the E-box/XRE and preventing interaction with the PR or PR-associated factors. As information from these studies becomes available, we will continue to explore the similarities (or differences) of TCDD modification of progesterone action versus RA action on MMP regulation.

As we develop a better understanding of the mechanisms of TCDD action in disrupting progesterone regulation of MMPs and cytokines, it becomes appropriate to begin in vivo studies. We will very shortly begin feeding studies in which mice are dosed with TCDD to determine the ability of progesterone to prevent or regress endometriosis. We anticipate that prior exposure to TCDD will greatly reduce the effects of progesterone action in this model.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 15 publications 9 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Bruner KL, Eisenberg E, Gorstein F, Osteen KG. Progesterone and transforming growth factor-β coordinately regulate suppression of endometrial matrix metalloproteinases in a model of experimental endometriosis. Steroids 1999;64(9):648-653. R826300 (1999)
R826300 (Final)
  • Abstract from PubMed
  • Abstract: ScinceDirect-Abstract
  • Journal Article Bruner-Tran KL, Rier SE, Eisenberg E, Osteen KG. The potential role of environmental toxins in the pathophysiology of endometriosis. Obstetric and Gynecologic Investigation 1999;48(Suppl 1):45-56. R826300 (1999)
    R826300 (Final)
  • Abstract from PubMed
  • Abstract: Karger-Abstract
  • Journal Article Osteen KG, Keller NR, Feltus FA, Melner MH. Paracrine regulation of matrix metalloproteinase expression in the normal human endometrium. Gynecologic and Obstetric Investigation 1999;48(Suppl 1):2-13. R826300 (1999)
    R826300 (Final)
  • Abstract from PubMed
  • Abstract: Karger-Abstract
  • Supplemental Keywords:

    dioxin, TCDD, progesterone action, animal model., RFA, Health, Scientific Discipline, Toxics, Environmental Chemistry, Health Risk Assessment, pesticides, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Children's Health, Biology, Endocrine Disruptors - Human Health, adverse outcomes, TCDD, dose response, steroid, enzymes, 2, 3, 7, 8-Tetrachloro-dibenzo-p-dioxin (TCDD), animal models, human exposure, toxicity, biochemistry, biological effects, dioxins, endometriosis, chlorinated phenols, ectopic growth, ovary

    Progress and Final Reports:

    Original Abstract
  • 1998
  • Final Report