Sakhila K. Banu

Selective Inhibition of Prostaglandin E2 Receptors EP2 and EP4 Induces Apoptosis of Human Endometriotic Cells through Suppression of ERK1/2, AKT, NFκB, and β-Catenin Pathways and Activation of Intrinsic Apoptotic Mechanisms

Endometriosis is a benign chronic gynecological disease of reproductive-age women characterized by the presence of functional endometrial tissues outside the uterine cavity. It is an estrogen-dependent disease. Current treatment modalities to inhibit biosynthesis and actions of estrogen compromise menstruation, pregnancy, and the reproductive health of women and fail to prevent reoccurrence of disease. There is a critical need to identify new specific signaling modules for non-estrogen-targeted therapies for endometriosis. In our previous study, we reported that selective inhibition of cyclooxygenase-2 prevented survival, migration, and invasion of human endometriotic epithelial and stromal cells, which was due to decreased prostaglandin E(2) (PGE(2)) production. In this study, we determined mechanisms through which PGE(2) promoted survival of human endometriotic cells. Results of the present study indicate that 1) PGE(2) promotes survival of human endometriotic cells through EP2 and EP4 receptors by activating ERK1/2, AKT, nuclear factor-kappaB, and beta-catenin signaling pathways; 2) selective inhibition of EP2 and EP4 suppresses these cell survival pathways and augments interactions between proapoptotic proteins (Bax and Bad) and antiapoptotic proteins (Bcl-2/Bcl-XL), facilitates the release of cytochrome c, and thus activates caspase-3/poly (ADP-ribose) polymerase-mediated intrinsic apoptotic pathways; and 3) these PGE(2) signaling components are more abundantly expressed in ectopic endometriosis tissues compared with eutopic endometrial tissues during the menstrual cycle in women. These novel findings may provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential therapy, including nonestrogen target, to expand the spectrum of currently available treatment options for endometriosis in women.

Molecular cloning and spatio-temporal expression of the prostaglandin transporter: A basis for the action of prostaglandins in the bovine reproductive system

Prostaglandins (PGs) play important roles in mammalian reproductive function through autocrine, paracrine, and endocrine actions. However, they predominate as charged anions and diffuse poorly across the plasma membrane. Recently, a PG transporter (PGT) has been found to mediate PG transport across cell membranes. In ruminants, endometrial PGs are transported by a vascular pathway to the ovary to regress or rescue the corpus luteum. There is no report on the role of PGT in the reproductive functions of any species. We have cloned and characterized the bovine PGT (bPGT) that transports different PGs in the following affinity order: PGE2 = PGF2α ≥ PGD2 much greater than arachidonate. bPGT mRNA and protein are expressed in endometrium, myometrium, and the utero-ovarian plexus (UOP) during the estrous cycle. The level of bPGT expression is higher in endometrium and UOP on the side of corpus luteum between days 13 and 18 of the estrous cycle. bPGT protein is localized in endometrial stroma, luminal epithelial cells, myometrial smooth muscle cells, and vascular smooth muscle cells of uterine vein and artery. In UOP, bPGT is selectively expressed in vascular smooth muscle cells of uterine vein and ovarian artery. Spatio-temporal expression of bPGT in uterine tissues and UOP supports a significant role of bPGT in cellular and compartmental transport of PGs to mediate the endocrine action at the time of luteolysis or establishment of pregnancy in bovine. This study describes and proposes a role of PGT in the regulation of reproductive processes.

Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits invasion of human immortalized endometriotic epithelial and stromal cells through suppression of metalloproteinases

Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. We recently reported that inhibition of COX-2 decreased migration as well as invasion of human endometriotic epithelial and stromal cells. Results of the present study indicates that selective inhibition of PGE2 receptors EP2 and EP4 suppresses expression and/or activity of MMP1, MMP2, MMP3, MMP7 and MMP9 proteins and increases expression of TIMP1, TIMP2, TIMP3, and TIMP4 proteins and thereby decreases migration and invasion of human immortalized endometriotic epithelial and stromal cells into matrigel. The interactions between EP2/EP4 and MMPs are mediated through Src and β-arrestin 1 protein complex involving MT1-MMP and EMMPRIN in human endometriotic cells. These novel findings provide an important molecular and cellular framework for further evaluation of selective inhibition of EP2 and EP4 as potential nonsteroidal therapy for endometriosis in childbearing-age women.

Hexavalent chromium-induced apoptosis of granulosa cells involves selective sub-cellular translocation of Bcl-2 members, ERK1/2 and p53

Hexavalent chromium (CrVI) has been widely used in industries throughout the world. Increased usage of CrVI and atmospheric emission of CrVI from catalytic converters of automobiles, and its improper disposal causes various health hazards including female infertility. Recently we have reported that lactational exposure to CrVI induced a delay/arrest in follicular development at the secondary follicular stage. In order to investigate the underlying mechanism, primary cultures of rat granulosa cells were treated with 10 μM potassium dichromate (CrVI) for 12 and 24h, with or without vitamin C pre-treatment for 24h. The effects of CrVI on intrinsic apoptotic pathway(s) were investigated. Our data indicated that CrVI: (i) induced DNA fragmentation and increased apoptosis, (ii) increased cytochrome c release from the mitochondria to cytosol, (iii) downregulated anti-apoptotic Bcl-2, Bcl-XL, HSP70 and HSP90; upregulated pro-apoptotic BAX and BAD, (iv) altered translocation of Bcl-2, Bcl-XL, BAX, BAD, HSP70 and HSP90 to the mitochondria, (v) upregulated p-ERK and p-JNK, and selectively translocated p-ERK to the mitochondria and nucleus, (vi) activated caspase-3 and PARP, and (vii) increased phosphorylation of p53 at ser-6, ser-9, ser-15, ser-20, ser-37, ser-46 and ser-392, increased p53 transcriptional activation, and downregulated MDM-2. Vitamin C pre-treatment mitigated CrVI effects on apoptosis and related pathways. Our study, for the first time provides a clear insight into the effect of CrVI on multiple pathways that lead to apoptosis of granulosa cells which could be mitigated by vitamin C.

Lactational exposure to hexavalent chromium delays puberty by impairing ovarian development, steroidogenesis and pituitary hormone synthesis in developing Wistar rats.

Hexavalent chromium (Cr-VI) is used in a wide range of industries. Cr-VI from chromate industries and atmospheric emissions contribute to the Cr contamination in the environment. Cr is a reproductive metal toxicant that can traverse the placental barrier and cause a wide range of fetal effects including ovotoxicity. Therefore, the goal of this study was to investigate the basic mechanisms involved in Cr(VI)-induced ovotoxicity, and the protective role of vitamin C on ovarian follicular development and function in Cr(VI)-induced reproductive toxicity using both in vivo and in vitro approaches. Lactating rats received potassium dichromate (200 mg/L) with or without vitamin C (500 mg/L), through drinking water from postpartum days 1-21. During postnatal days (PND) 1-21 the pups received Cr(VI) via the mothers milk. Pups from both control and treatment groups were continued on regular diet and water from PND-21 onwards, and euthanized on PND-21, -45 and -65. Cr(VI) decreased steroidogenesis, GH and PRL, increased FSH and did not alter LH. Cr(VI) delayed puberty, decreased follicle number, and extended estrous cycle. Spontaneously immortalized rat granulosa cells were treated with 12.5 microM (IC(50)) potassium dichromate for 12 and 24 h, with or without vitamin C pre-treatment. Cr(VI) decreased the mRNA expressions of StAR, SF-1, 17beta-HSD-1, 17beta-HSD-2, FSHR, LHR, ER alpha and ER beta. Vitamin C pre-treatment protected ovary and granulosa cells from the deleterious effects of Cr(VI) toxicity, both in vivo and in vitro. Therefore, Cr(VI) toxicity could be a potential risk to the reproductive system in developing females, and vitamin C plays a protective role against Cr(VI)-induced ovotoxicity.

Transport of Prostaglandin F2α Pulses from the Uterus to the Ovary at the Time of Luteolysis in Ruminants Is Regulated by Prostaglandin Transporter-Mediated Mechanisms

In ruminants, prostaglandin F2alpha (PGF(2alpha)) is the uterine luteolytic hormone. During luteolysis, PGF(2alpha) is synthesized and released from the endometrium in a pulsatile pattern. The unique structure of the vascular utero-ovarian plexus (UOP) allows transport of luteolytic PGF(2alpha) pulses directly from the uterus to the ovary, thus bypassing the systemic circulation. However, the underlying molecular mechanism is not known. The objective of the present study was to determine a role for PG transporter protein (PGT) in the compartmental transport of PGF(2alpha) from uterus to ovary through the UOP at the time of luteolysis using the sheep as a ruminant model. [(3)H]PGF(2alpha), with or without a PGT inhibitor, was infused into UOP, and PGF(2alpha) transport and PGT protein expression were determined. Results indicate that PGT protein is expressed in tunica intima, tunica media, and tunica adventitia of the utero-ovarian vein and the ovarian artery of the UOP, and the expression levels are higher on d 10-15 compared with d 3-6 of the estrous cycle. Pharmacological inhibition of PGT prevented transport of exogenous [(3)H]PGF(2alpha) as well as oxytocin-induced endogenous luteolytic PGF(2alpha) pulse up to 80% from uterine venous blood into ovarian arterial blood through the UOP at the time of luteolysis in sheep. Taken together, these results indicate that at the time of luteolysis, transport of PGF(2alpha) from uterus to ovary through the UOP is regulated by PGT-mediated mechanisms. These findings also suggest that impaired PGT-mediated transport of PGF(2alpha) from the utero-ovarian vein into the ovarian artery could adversely influence luteolysis and thus affect fertility in ruminants.

Intraluteal Prostaglandin Biosynthesis and Signaling Are Selectively Directed Towards PGF2alpha During Luteolysis but Towards PGE2 During the Establishment of Pregnancy in Sheep

ABSTRACT In ruminants, endometrial prostalgandin (PG) F2alpha causes functional luteolysis, whereas luteal synthesis of PGF2alpha is required for structural luteolysis. PGE2 is considered to be a luteoprotective mediator. Molecular aspects of luteal PGF2alpha and PGE2 biosynthesis and signaling during the estrous cycle and establishment of pregnancy are largely unknown. The objectives of the present study were 1) to determine the regulation of proteins involved in PGF2alpha and PGE2 biosynthesis, catabolism, transport and signaling in the corpus luteum (CL); 2) to investigate the transport of interferon tau (IFNT), PGF2alpha, and PGE2 from the uterus to the ovary through the vascular utero-ovarian plexus (UOP); and 3) to compare the intraluteal production of PGF2alpha and PGE2 on Days 12, 14, and 16 of the estrous cycle and pregnancy in sheep. Our results indicate that luteal PG biosynthesis is selectively directed towards PGF2alpha at the time of luteolysis and towards PGE2 during the establishment of pregnancy. Moreover, the ability of the CL of early pregnancy to resist luteolysis is due to increased intraluteal biosynthesis of PGE2 and PGE2 receptor (PTGER) 2 (also known as EP2)- and PTGER4 (also known as EP4)-mediated signaling. We also found that IFNT protein is not transported through the UOP from the uterus to the ovary; in contrast, a large proportion of endometrial PGE2 is transported from the uterus to the ovary through the UOP. These results indicate that endometrial PGE2 stimulated by pregnancy is transported locally to the ovary, which increases luteal PGE2 biosynthesis and hence activates luteal PTGER2 and PTGER4 signaling, thus protecting the CL during the establishment of pregnancy in sheep.

Interferon Tau Regulates PGF2α Release from the Ovine Endometrial Epithelial Cells via Activation of Novel JAK/EGFR/ERK/EGR-1 Pathways

In ruminants, pulsatile release of prostaglandin F2α (PGF(2α)) from the endometrium is transported to the ovary and induces luteolysis thereby allowing new estrous cycle. Interferon tau (IFNT), a type 1 IFN secreted by the trophoblast cells of the developing conceptus, acts on endometrial luminal epithelial (LE) cells and inhibits pulsatile release of PGF(2α) and establishes pregnancy. One of the unknown mechanisms is that endometrial pulsatile release of PGF(2α) is inhibited whereas basal release of PGF(2α) is increased in pregnant compared with nonpregnant sheep. We have recently found that pulsatile release of PGF(2α) from the endometrium is regulated by prostaglandin transporter (PGT)-mediated mechanisms. We hypothesize that modulation in the endometrial pulsatile vs. basal release of PGF(2α) likely requires PGT-mediated selective transport, and IFNT interacts with PGT protein and modulates pulsatile vs. basal release of PGF(2α). The new findings of the present study are: 1) IFNT activates novel JAK-SRC kinase-EGFR-RAS-RAF-ERK1/2-early growth response (EGR)-1 signaling module in LE cells; 2) IFNT increases interactions between PGT and ERK1/2 or EGR-1 proteins and alters phosphorylation of PGT protein; 3) IFNT precludes action of protein kinase C and Ca(2+) on PGT function; and 4) IFNT inhibits 80% PGT-mediated but not 20% simple diffusion-mediated release of PGF(2α) from the endometrial LE cells through this novel signaling module. The results of the present study provide important new insights on IFNT signaling and molecular control of PGT-mediated release of PGF(2α) and unravel the underlying mechanisms responsible for the increased basal release of PGF(2α) at the time of establishment of pregnancy in ruminants.

Early pregnancy induced expression of prostaglandin E2 receptors EP2 and EP4 in the ovine endometrium and regulated by interferon tau through multiple cell signaling pathways

Prostaglandin E2 (PGE(2)) plays pleiotropic roles at fetal-maternal interface during establishment of pregnancy. The objectives of the study were to: (i) determine regulation of PGE2 receptors EP1, EP2, EP3, and EP4 in the endometrium during the estrous cycle and early pregnancy; and (ii) understand endometrial epithelial and stromal cell-specific hormonal regulation of EP2 and EP4 in sheep. Results indicate that: (i) early pregnancy induces expression of EP2 and EP4 but not EP1 and EP3 proteins in the endometrium on days 12-16 compared to that of estrous cycle; (ii) intrauterine infusion of interferon tau (IFNT) increases expression of EP2 and EP4 proteins in endometrium; and (iii) IFNT activates distinct epithelial and stromal cell-specific JAK, EGFR, ERK1/2, AKT, or JNK signaling module to regulate expression of EP2 and EP4 proteins in the ovine endometrium. Our results indicate a role for EP2 and EP4-mediated PGE(2) signaling in endometrial functions and establishment of pregnancy in ruminants.

Induction of peritoneal endometriosis in nude mice with use of human immortalized endometriosis epithelial and stromal cells: a potential experimental tool to study molecular pathogenesis of endometriosis in humans

OBJECTIVE To determine whether a mixed population of immortalized human endometriosis epithelial and stromal cells is able to induce peritoneal endometriosis in nude mice. DESIGN Prospective experimental study. Human immortalized endometriosis epithelial and stromal cells were xenografted into ovariectomized nude mice. Macroscopically, the number of induced endometriosis-like lesions and their color were determined. Microscopically, histomorphology of endometriosis glands and their structure were analyzed, and comparisons were made with tissue from spontaneous endometriosis in women. SETTING College of Veterinary Medicine and Biomedical Sciences, Texas A&M University. ANIMALS Seven ovariectomized nude mice. INTERVENTION(S) Minimal invasive procedures were performed to administer estrogen pellets and transplant immortalized human endometriosis epithelial and stromal cells into nude mice. MAIN OUTCOME MEASURE(S) Peritoneal endometriosis-like lesions induced in nude mice were characterized and compared with spontaneous peritoneal endometriosis in women. RESULT(S) Xenografts of human immortalized endometriosis epithelial and stromal cells into the peritoneal cavity of the recipient nude mice are able to proliferate, attach, invade, reorganize, and establish peritoneal endometriosis. Endometriosis glands at different stages of growth were present in induced endometriosis-like lesions. Proliferating cell nuclear antigen, metalloproteinase 2, estrogen receptor-alpha, cyclooxygenase-2, and prostaglandin E(2) receptors EP2 and EP4 proteins were expressed in both endometriosis glandular epithelial and stromal cells of the induced endometriosis-like lesions. CONCLUSION(S) This xenograft model could be used as a potential experimental tool to understand the molecular and cellular aspects of the pathogenesis of endometriosis in humans.