Regulation of phosphatidylinositide turnover in the myometrium by cAMP: Implications for the control of uterine contraction

Regulation of uterine quiescence involves the integration of the signaling pathways regulating uterine contraction and relaxation. Uterine contractants increase intracellular calcium through receptor/GαqPLC coupling, resulting in contraction of the myometrium. Elevation of cAMP concentration has been correlated with relaxation of the myometrium. However, the mechanism of cAMP action in the uterus is unclear. ^ Both endogenous and exogenous increases in cAMP inhibited oxytocin-stimulated phosphatidylinositide turnover in an immortalized pregnant human myometrial cell line (PHM1-41). This inhibition was reversed by cAMP-dependent protein kinase (PKA) inhibitors, suggesting the involvement of PKA. cAMP inhibited phosphatidyinositide turnover stimulated by different agonists in different cell lines. These data suggest that the cAMP inhibitory mechanism is neither cell nor receptor dependent, and inhibits Gαq/PLCβ1 and PLCβ3 coupling. ^ The subcellular localization of PKA occurs via PKA binding to A-Kinase-Anchoring-Proteins (AKAP), and peptides that inhibit this association have been developed (S-Ht31). S-Ht31 blocked cAMP-stimulated PKA activity and decreased PKA concentration in PHM1-41 cell plasma membranes. S-Ht31 reversed the ability of CPT-cAMP, forskolin and relaxin to inhibit phosphatidylinositide turnover in PHM1-41 cells. Overlay analysis of both PHM1-41 cell and nonpregnant rat myometrium found an AKAPs of 86 kDa and 150 kDa associated with the plasma membrane, respectively. These data suggest that PKA anchored to the plasma membrane via AKAP150/PKA anchoring is involved in the cAMP inhibitory mechanism. ^ CPT-cAMP and isoproterenol inhibited phosphatidylinositide turnover in rat myometrium from days 12 through 20 of gestation. In contrast, neither agent was effective in the 21 day pregnant rat myometrium. The decrease in the cAMP inhibitory mechanism was correlated with a decrease in PKA and an increase in protein phosphatase 2B (PP2B) concentration in rat myometrial plasma membranes on day 21 of gestation. In myometrial total cell homogenates, both PKA and PP2B concentration increased on day 21. S-Ht31 inhibited cAMP inhibition of phosphatidylinositide turnover in day 19 pregnant rat myometrium. Both PKA and PP2B coimmunoprecipitated with an AKAP150 in a gestational dependent manner, suggesting this AKAP localizes PKA and PP2B to the plasma membrane. ^ These data presented demonstrate the importance of the cAMP inhibitory mechanism in regulating uterine contractility. ^

XENOESTROGEN-SPECIFIC MECHANISMS OF DEVELOPMENTAL REPROGRAMMING CORRELATE WITH GENE EXPRESSION AND TUMOR DEVELOPMENT

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. We have shown that neonatal exposure to the xenoestrogen diethylstilbestrol (DES) can developmentally reprogram the reproductive tract in genetically susceptible Eker rats giving rise to complete penetrance of uterine leiomyoma. Based on this, we hypothesized that xenoestrogens, including genistein (GEN) and bisphenol A (BPA), reprogram estrogen-responsive gene expression in the myometrium and promote the development of uterine leiomyoma. We proposed the mechanism that is responsible for the developmental reprogramming of gene expression was through estrogen (E2)/ xenoestrogen inducedrapid ER signaling, which modifies the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2) via activation of the PI3K/AKT pathway. We further hypothesized that there is a xenostrogen-specific effect on this pathway altering patterns of histone modification, DNA methylation and gene expression. In addition to our novel finding that E2/DES-induced phosphorylation of EZH2 by AKT reduces the levels of H3K27me3 in vitro and in vivo, this work demonstrates in vivo that a brief neonatal exposure to GEN, in contrast to BPA, activates the PI3K/AKT pathway to regulate EZH2 and decreases H3K27me3 levels in the neonatal uterus. Given that H3K27me3 is a repressive mark that has been shown to result in DNA methylation and gene silencing we investigated the methylation of developmentally reprogrammed genes. In support of this evidence, we show that neonatal DES exposure in comparison to VEH, leads to hypomethylation of the promoter of a developmentally reprogrammed gene, Gria2, that become hyper-responsive to estrogen in the adult myometrium indicating vi that DES exposure alter gene expression via chromatin remodeling and loss of DNA methylation. In the adult uterus, GEN and BPA exposure developmentally reprogrammed expression of estrogen-responsive genes in a manner opposite of one another, correlating with our previous data. Furthermore, the ability of GEN and BPA to developmental reprogram gene expression correlated with tumor incidence and multiplicity. These data show that xenoestrogens have unique effects on the activation of non-genomic signaling in the developing uterus that promotes epigenetic and genetic alterations, which are predictive of developmental reprogramming and correlate with their ability to modulate hormone-dependent tumor development.

Dicer is required for female reproductive tract development and fertility in the mouse.

Dicer encodes a riboendonuclease required for microRNA biosynthesis. Dicer was inactivated in Müllerian duct mesenchyme-derived tissues of the reproductive tract of the mouse, using an Amhr2-Cre allele. Although Amhr2-Cre; Dicer conditional mutant males appeared normal and were fertile, mutant females were infertile. In adult mutant females, there was a reduction in the size of the oviducts and uterine horns. The oviducts were less coiled compared to controls and cysts formed at the isthmus near the uterotubal junction. Unfertilized, degenerate oocytes were commonly found within these cysts, indicating a defect in embryo transit. Beads transferred into the mutant oviduct failed to migrate into the uterus. In addition, blastocysts transferred directly into the mutant uterus did not result in pregnancy. Histological analysis demonstrated that the mutant uterus contained less glandular tissue and often the few glands that remained were found within the myometrium, an abnormal condition known as adenomyosis. In adult mutants, there was ectopic expression of Wnt4 and Wnt5a in the luminal epithelium (LE) and glandular epithelium (GE) of the uterus, and Wnt11 was ectopically expressed in GE. These results demonstrate that Dicer is necessary for postnatal differentiation of Müllerian duct mesenchyme-derived tissues of the female reproductive tract, suggesting that microRNAs are important regulators of female reproductive tract development and fertility.

THE EFFECT OF RELAXIN ON BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS OF RAT MYOMETRIAL CELLS IN CULTURE (CYCLIC-AMP, CELL SHAPE CHANGE)

Relaxin is able to inhibit spontaneous, oxytocin-and prostaglandin-driven uterine contractions. The intracellular mechanism of action of relaxin on uterine relaxation had previously been studied using isometrically suspended uterine strips. Since uterine strips contain stroma as well as myometrium, the changes in biochemical parameters induced by relaxin treatment may not occur in the same cell types responsible for the physical changes. In these studies, cultures of enriched populations of rat myometrial cells were used to investigate the effect of relaxin on biochemical and morphological parameters which are related to relaxation.^ Under optimal culture conditions (initial plating density 1 - 1.5 x 10('6)cells/ml, 3 ml/35 mm dish, 2 days culture), enzymatically isolated rat myometrial cells were able to respond to relaxin with cAMP elevation. Relaxin elevated cAMP levels in the presence but not the absence of 0.1 mM methylisobutylxanthine or 0.4 um forskolin in a time- and concentration-dependent manner. In contrast, isoproterenol was able to elevate cAMP levels in the presence and absence of 0.1 mM methylisobutylxanthine.^ Oxytocin treatment caused a decrease in mean cell length and area of myometrial cells in culture which could be considered analogous to contraction. Under optimal culture conditions, relaxin increased myometrial cell length and area (i.e. analogous to relaxation) of oxytocin-treated cells in a time- and concentration-dependent manner. Other relaxants such as isoproterenol and dibutyryl cAMP also increased cell length and area of oxytocin - treated myometrial cells in culture.^ Under optimal culture conditions, relaxin decreased myosin light chain kinase activity in a time-and concentration-dependent manner by increasing the K(,50) of the enzyme for calmodulin (CaM), i.e. decreasing the affinity of the enzyme for CaM. The decrease in the affinity of myosin light chain kinase for CaM may be due to the phosphorylation of the enzyme by cAMP-dependent protein kinase. Relaxin also decreased the Ca('2+)(.)CaM-independent myosin light chain kinase activity to a lesser extent than that of the Ca('2+)(.)CaM-dependent enzyme activity. This was not attributable to a decrease in the affinity of the enzyme for myosin in myometrial cells in culture, in contrast to the finding of such a change following relaxin treatment of uterine strips. Further studies are required to clarify this point.^ There was a temporal association between the effects of relaxin on elevation of cAMP levels in the presence of 0.4 uM forskolin, increase in cell length and decrease in myosin light chain kinase activity. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Convergent etiology of Eker rat and human uterine leiomyoma

The Eker rat model has allowed researchers the unique opportunity to study the tumorigenesis of spontaneously occurring uterine leiomyoma. Animals in this line harbor a germline mutation in the tuberous sclerosis complex-2 (Tsc-2) tumor suppressor gene and develop uterine leiomyomas at a rate of ∼65%. Primary leiomyomas obtained from humans and Eker rats along with Eker-derived leiomyoma cell lines were used in studies described herein to determine the effect of PPARγ ligand treatment on the proliferation of this cell type and to determine the role of tuberin and p27Kip1 in the etiology of this tumor type. Treatment of leiomyoma cells of human and rat origin with PPARγ-activating compounds resulted in decreased proliferation. Additionally, PPARγ ligands inhibited estrogen-dependent gene transactivation in Eker-derived leiomyoma cells suggesting that nuclear receptor cross-talk may exist between PPAR and the ER and may be responsible for the inhibition of proliferation in this cell type. Loss of tuberin, the product of the TSC-2 gene, is associated with Eker rat leiomyoma development while the role of this tumor suppressor in human leiomyoma development is unknown. Data herein show that tuberin expression is diminished in 25% of human leiomyomas tested. Additionally, we observed diminished p27 Kip1 expression in 80% of human uterine leiomyomas compared to normal myometrium. Interestingly, the loss of tuberin expression in human leiomyoma was associated with cytoplasmic p27Kip1 accumulation in this cell type. Furthermore, tuberin-null Eker rat leiomyomas and derived cell lines had predominantly cytoplasmic p27Kip1 compared to tuberin-expressing normal myometrium. Taken together, our data show that human and Eker rat leiomyoma proliferation is inhibited upon PPARγ treatment and that the etiology of human and Eker rat leiomyoma converge at loss of p27Kip1 function. Furthermore, our data indicate that the loss of p27 Kip1 function is mediated by loss of expression (in 80% of human leiomyoma) or cytoplasmic localization potentially resulting from the loss of tuberin. ^

Developmental programming of tumor suppressor gene penetrance

Epidemiological studies have led to the hypothesis that major risk factors for developing diseases such as hypertension, cardiovascular disease and adult-onset diabetes are established during development. This developmental programming hypothesis proposes that exposure to an adverse stimulus or insult at critical, sensitive periods of development can induce permanent alterations in normal physiological processes that lead to increased disease risk later in life. For cancer, inheritance of a tumor suppressor gene defect confers a high relative risk for disease development. However, these defects are rarely 100% penetrant. Traditionally, gene-environment interactions are thought to contribute to the penetrance of tumor suppressor gene defects by facilitating or inhibiting the acquisition of additional somatic mutations required for tumorigenesis. The studies presented herein identify developmental programming as a distinctive type of gene-environment interaction that can enhance the penetrance of a tumor suppressor gene defect in adult life. Using rats predisposed to uterine leiomyoma due to a germ-line defect in one allele of the tuberous sclerosis complex 2 (Tsc-2) tumor suppressor gene, these studies show that early-life exposure to the xenoestrogen, diethylstilbestrol (DES), during development of the uterus increased tumor incidence, multiplicity and size in genetically predisposed animals, but failed to induce tumors in wild-type rats. Uterine leiomyomas are ovarian-hormone dependent tumors that develop from the uterine myometrium. DES exposure was shown to developmentally program the myometrium, causing increased expression of estrogen-responsive genes prior to the onset of tumors. Loss of function of the normal Tsc-2 allele remained the rate-limiting event for tumorigenesis; however, tumors that developed in exposed animals displayed an enhanced proliferative response to ovarian steroid hormones relative to tumors that developed in unexposed animals. Furthermore, the studies presented herein identify developmental periods during which target tissues are maximally susceptible to developmental programming. These data suggest that exposure to environmental factors during critical periods of development can permanently alter normal physiological tissue responses and thus lead to increased disease risk in genetically susceptible individuals. ^

ROLE OF SOX9 IN UTERINE GLAND DEVELOPMENT AND DISEASE INITIATION

The female reproductive tract (FRT) develops midway through embryogenesis, and consists of oviducts, uterine horns, cervix and upper part of the vagina. The uterine horns are composed of an epithelial layer, luminal (LE) and glandular epithelium (GE), surrounded by a mesenchymal layer, the stroma and myometrium. Interestingly, in most mammals the GE forms after birth and it only becomes fully differentiated as the female reaches sexual maturity. Uterine glands (UG) are made up of GE and are present in all mammals. They secrete nutrients, cytokines and several other proteins, termed histotroph, that are necessary for embryo implantation and development. Experiments in ewes and mice have revealed that females who lack UGs are infertile mainly due to impaired implantation and early pregnancy loss, suggesting that UGs are essential for fertility. Fortunately for us, UGs develop after birth allowing us to peer into the genetic mechanism of tubulogenesis and branching morphogenesis; two processes that are disrupted in various adenocarcinomas (cancer derived from glands). We created 3D replicas of the epithelium lining the FRT using optical projection tomography and characterized UG development in mice using lineagetracing experiments. Our findings indicate that mouse UGs develop as simple tubular structures and later grow multiple secretory units that stem from the main duct. The main aim of this project was to study the role of SOX9 in the UGs. Preliminary studies revealed that Sox9 is mostly found in the nucleus of the GE. vii This observation led to the hypothesis that Sox9 plays a role in the formation and/or differentiation of the GE. To study the role of Sox9 in UGs differentiation, we conditionally knocked out and overexpressed Sox9 in both the LE and GE using the progesterone receptor (Pgr) promoter. Overexpressing Sox9 in the uterine epithelium, parts of the stroma, and myometrium led to formation of multiple cystic structures inside the endometrium. Histological analysis revealed that these structures appeared morphologically similar to structures present in histological tissue sections obtained from patients with endometrial polyps. We have accounted for the presence of simple and complex hyperplasia with atypia, metaplasia, thick-walled blood vessels, and stromal fibrosis; all “hallmarks” that indicate overexpressing Sox9 leads to development of a polyp-like morphology. Therefore, we can propose the use of Sox9-cOE mice to study development of endometrial cystic lesions and disease progression into hyperplastic lesions.

Identifying and Targeting Molecular Deregulations in Uterine Leiomyosarcoma: A Role for mTOR Inhibition-Based Combination Therapies

Uterine leiomyosarcoma (ULMS) is an aggressive malignancy characterized by marked chemoresistance, frequent relapses, and poor outcome. Despite efforts to improve survival over the past several decades, only minimal advances have been made. Hence, there is an urgent and unmet need for better understanding of the molecular deregulations that underlay ULMS and development of more effective therapeutic strategies. This work identified several common deregulations in a large (n=208) tissue microarray of ULMS compared to GI smooth muscle, myometrium, and leiomyoma controls. Our results suggest that significant loss of smooth muscle and gynecological differentiation markers is common in ULMS, a finding that could help render improved ULMS diagnosis, especially for advanced disease. Similarly to reports in other malignancies, we found that several cancer-related proteins were differentially expressed; these could be useful together as biomarkers for ULMS. Notably, we identified significant upregulation and overexpression of the mTOR pathway in ULMS, examined the possible contribution of tyrosine kinase receptor deregulation promoting mTOR activation, and unraveled a role for pS6RP and p4EBP1 as molecular disease prognosticators. The significance of mTOR activation in ULMS and its potential as a therapeutic target were further investigated. Rapamycin abrogated ULMS cell growth and cell cycle progression in vitro but induced only sight growth delay in vivo. Given that effective mTOR therapies likely require combination mTOR blockade with inhibition of other targets, coupled with recent observations suggesting that Aurora A kinase (Aurk A) deregulations commonly occur in ULMS, the preclinical impact of dually targeting both pathways was evaluated. Combined therapy with rapamycin (an mTORC1 inhibitor) and MLN8237 (an investigational Aurk A inhibitor) profoundly and synergistically abrogated ULMS growth in vitro. Interestingly, the superior effects were noted only when MLN8237 was pre-administered. This novel therapeutic combination and scheduling regimen resulted in marked tumor growth inhibition in vivo. Together, these data support further exploration of dual mTOR and Aurk A blockade for the treatment of human ULMS.

Critical determinants of estrogen receptor -mediated agonist activity

Exogenous ligands that bind to the estrogen receptor (ER) exhibit unique pharmacologies distinct from that observed with the endogenous hormone, 17β-estradiol (ED. Differential activity among ER ligands has been observed at the level of receptor binding, promoter interaction and transcriptional activation. Furthermore, xenoestrogens can display tissue-specific agonist activity on the cellular level, functioning as an agonist in one tissue and as an antagonist in another. That the same ligand, functioning through the same receptor, can produce differing agonist responses on the cellular level indicates that there are tissue-specific determinants of agonist activity. In these studies critical molecular determinants of agonist activity were characterized for several cell types. In the normal and neoplastic myometrium a proliferative response was dependent upon activation of AF2 of the ER, functioning as a determinant of agonism in this cell type. Progesterone receptor (PR) ligands transdominantly suppressed ER-mediated transcription and proliferation in uterine leiomyoma cells, indicating that ER/PR cross-talk can modulate agonist activity in a myometrial cell background. In the breast, the agonist response to ER ligands was investigated by employing a functional genomics approach to generate gene expression profiles. Treatment of breast cancer cells with the selective estrogen receptor modulator tamoxifen largely recapitulated the expression profile induced by treatment with the agonist E2, despite the well-characterized antiproliferative effects produced by tamoxifen in this cell type. While the expression of many genes involved in regulating cell cycle progression, including fos, myc, cdc25a, stk15 and cyclin A, were induced by both E2 and tamoxifen in breast cells, treatment with the agonist E2 specifically induced the expression of cyclin D1, fra-1 , and uracil DNA glycosylase. These results suggest that the inability of tamoxifen to transactivate expression of only a few key genes, functioning as cellular gatekeepers, prevent tamoxifen-treated breast cells from entering the cell cycle. Thus, the expression of these agonist-specific marker genes is a potential determinant of agonist activity at the cellular level in the breast. Collectively, studies in the breast and uterine myometrium have identified several mechanisms whereby ER ligands modulate ER-mediated signaling and provide insights into the biology of tissue-specific agonist activity in hormone-responsive tissues. ^