The contribution of a REST-MMP tissue remodeling pathway to adenomyosis pathophysiology

Adenomyosis is a non-neoplastic uterine disease characterized by the invasion and migration of endometrial glands and stroma into the myometrial layer of the uterus. Adenomyosis is suspected to have a substantial presence globally, affecting anywhere between 20-65% of pre-menopausal women. The development of adenomyosis is often correlated with severe symptomology that negatively affects the quality of life of these individuals. Severe symptomology includes chronic pelvic pain, dysmenorrhea, menorrhagia, abnormal uterine bleeding, and secondary infertility. A popular developmental theory of adenomyosis is the tissue injury and remodeling theory, known as TIAR. This theory postulates the involvement of tissue remodeling mechanisms and wound healing pathways like the epithelial to mesenchymal transition (EMT) which are further amplified by progesterone resistance, which is characteristic of the disease. The increase of tissue remodeling pathways indicates a disruption in the tight regulation of tissue remodeling proteins like matrix metalloproteinases (MMPs). MMPs function to breakdown the extracellular matrix (ECM), which is a critical component in the destruction of the endometrial functionalis during menses. MMPs are suppressed by the steroid hormone, progesterone. RE-1 silencing transcription factor, REST, is a transcriptional repressor which has been shown to function as a tumor suppressor and, consequently, significantly reduced in association with progesterone resistance in breast cancer and endometrial cancer. Based on the evidence outlined above, our lab, in collaboration with Dr. Elizabeth Stewart at the Obstetrics and Gynecology Department at the Mayo Clinic, investigated the potential contribution of a REST-MMP tissue remodeling pathway to adenomyosis pathophysiology. To assess the involvement of a REST-MMP tissue remodeling pathway, we assessed potential protein misexpression using human uterine biopsies. We found that during the secretory phase of the menstrual cycle, REST is significantly reduced and is associated with a reduction in the expression of the extracellular matrix-bound MMP inhibitor, RECK, which allows for a rise in MMP-9 expression levels. Using control uterine biopsies, we isolated endometrial stromal cells to assess changes in phenotypic characteristics based on a reduction of REST. While we did not observe a significant change in proliferation rates, we did observe an increase in cell migration when REST is reduced in isolated human endometrial stromal cells. In order to assess risk factors often associated with increased adenomyosis incidence rates, our lab created a novel conditional knockout mouse model using a progesterone receptor Cre-lox system. This allowed for the conditional knockout of Rest in the endometrium and myometrium of the uterus. Using this mouse model, we assessed the involvement of the hypothesized REST-MMP tissue remodeling pathway in correlation to aging, and uterine trauma by uterine surgery and increased parity. In collaboration with Dr. Kaylon Bruner-Tran’s laboratory from Vanderbilt University, we were able to assess the hypothesized REST-MMP cascade in correlation to in-utero exposure of dioxin. In our assessment of age, we found that regardless of age, 6-, 9-, and 12-months, Mmp-24 is significantly increased in female Rest cKO mice compared to female control mice. At 12-months of age, Rest cKO mice demonstrated significant reduction of RECK compared to control mice. However, Mmp-2 and Mmp-9 transcript were unchanged in all age groups. However, using zymography analysis we found that while MMP-9 protein and mRNA levels were not elevated, activity levels were significantly elevated in Rest cKO mice compared to control mice. Next, we assessed the activity of the REST-MMP cascade when exposed to dioxin in-utero. We found that MMP-24 and MMP-9 expression was both elevated in the myometrium of F1 generation dioxin exposed mice compared to controls. In the F2 generation, MMP-24 maintained elevated levels in the myometrium and MMP-2 expression was increased in stromal cells in the dioxin exposed mice compared to control mice. Finally, in our assessment of uterine trauma, we observed the involvement of the REST-MMP tissue remodeling cascade in the traumatized uterine horn of control mice. We also saw the development of adenomyosis regardless of genotype in traumatized uterine horns. When assessing the effects of increased parity, we observed an increased incident rate of adenomyosis in Rest cKO mice compared to control mice. Our study suggests the involvement of the hypothesized REST-MMP tissue remodeling cascade, contributing to the pathophysiology of adenomyosis.