Abstract: Many signaling pathways that contribute to tumorigenesis are also functional in pregnancy, although they are dysregulated in the former and tightly regulated in the latter. Transformation-related protein 53 (Trp53), which encodes p53, is a tumor suppressor gene whose mutation is strongly associated with cancer. However, its role in normal physiological processes, including female reproduction, is poorly understood. Mice that have a constitutive deletion of Trp53 exhibit widespread development of carcinogenesis at early reproductive ages, compromised spermatogenesis, and fetal exencephaly, rendering them less amenable to studying the role of p53 in reproduction. To overcome this obstacle, we generated mice that harbor a conditional deletion of uterine Trp53 and examined pregnancy outcome in females with this genotype. These mice had normal ovulation, fertilization, and implantation; however, postimplantation uterine decidual cells showed terminal differentiation and senescence-associated growth restriction with increased levels of phosphorylated Akt and p21, factors that are both known to participate in these processes in other systems. Strikingly, uterine deletion of Trp53 increased the incidence of preterm birth, a condition that was corrected by oral administration of the selective COX2 inhibitor celecoxib. We further generated evidence to suggest that deletion of uterine Trp53 induces preterm birth through a COX2/PGF synthase/PGF(2alpha) pathway. Taken together, our observations underscore what we believe to be a new critical role of uterine p53 in parturition.
Abstract: Immunophilin FK506-binding protein 52 (FKBP52) is a cochaperone that binds to the progesterone receptor (PR) to optimize progesterone (P(4))-PR signaling. We recently showed that Fkbp52-deficient (Fkbp52(-/-)) mice have reduced uterine PR responsiveness and implantation failure which is rescued by excess P(4) supplementation in a genetic background-dependent manner. This finding led us to hypothesize that FKBP52 has functions in addition to optimizing PR activity. Using proteomics analysis, we found that uterine levels of peroxiredoxin-6 (PRDX6), a unique antioxidant, are significantly lower in Fkbp52(-/-) mice than in WT and PR-null (Pgr(-/-)) mice. We also found that Fkbp52(-/-) mice with reduced uterine PRDX6 levels are susceptible to paraquat-induced oxidative stress (OS), leading to implantation failure even with P(4) supplementation. The same dose of paraquat did not interfere with implantation in WT mice. Moreover, treatment with antioxidants alpha-tocopherol and N-acetylcysteine (NAC) attenuated paraquat-induced implantation failure in P(4)-treated Fkbp52(-/-) mice. Functional analyses using mouse embryonic fibroblasts show that Fkbp52 deficiency associated with reduced PRDX6 levels promotes H(2)O(2)-induced cell death, which is reversed by the addition of NAC or by forced expression of PRDX6, suggesting that Fkbp52 deficiency diminishes the threshold against OS by reducing PRDX6 levels. These findings provide evidence that heightened uterine OS in Fkbp52(-/-) females with reduced PRDX6 levels induces implantation failure even in the presence of excess P(4). This study shows that FKBP52-PRDX6 signaling protects pregnancy from overt OS.
Abstract: Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI Imaging Mass Spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4-8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A2a and cyclooxygenase-2 during embryo implantation. Liquid chromatography-electrospray ionization tandem MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.
Abstract: The activation of the blastocyst, a process by which it gains competency to attach with the receptive uterus, is a prerequisite for successful implantation. However, the molecular basis of blastocyst activation remains largely unexplored. Combining molecular, pharmacological and physiological approaches, we show here that silencing of Wnt-beta-catenin signaling in mice does not adversely affect the development of preimplantation embryos to blastocysts and uterine preparation for receptivity, but, remarkably, blocks blastocyst competency to implantation. Using the physiologically relevant delayed implantation model and trophoblast stem cells in culture, we further demonstrate that a coordinated activation of canonical Wnt-beta-catenin signaling with attenuation of the non-canonical Wnt-RhoA signaling pathway ensures blastocyst competency to implantation. These findings constitute novel evidence that Wnt signaling is at least one pathway that determines blastocyst competency for implantation.
Abstract: A reciprocal interaction between the implantation-competent blastocyst and receptive uterus is an absolute requirement for implantation, a process crucial for pregnancy success. A comprehensive understanding of this interaction has yet to be realized. One major difficulty in clearly defining this discourse is the complexity of the implantation process involving heterogeneous cell types of both the uterus and blastocyst, each endowed with unique molecular signatures that show dynamic changes during the course of pregnancy. Whereas gene expression studies by in situ hybridization or immunohistochemistry have shown differential expression patterns of specific genes during implantation, there is no report how numerous signaling proteins are spatially displayed at specific times and stages of implantation in the context of blastocyst-uterine juxtaposition. Using in situ imaging (matrix assisted laser desorption/ionization) mass spectrometry directly on uterine sections, here we provide molecular composition, relative abundance, and spatial distribution of a large number of proteins during the periimplantation period. This approach has allowed us for the first time to generate in situ proteome profiles of implantation and interimplantation sites in mice in a region- and stage-specific manner with the progression of implantation. This application is reliable because patterns of expression of several proteins displayed by in situ imaging mass spectrometry correlate well with in situ hybridization results. More interestingly, the use of this approach has provided new insights regarding uterine biology of cytosolic phospholipase A(2alpha) null females that show implantation defects.
Abstract: Endometriosis is a common gynecological disease that affects approximately 10% of women of childbearing age. It is characterized by endometrial growth outside the uterus and often results in inflamed lesions, pain, and reduced fertility. Although heightened estrogenic activity and/or reduced progesterone responsiveness are considered to be involved in the etiology of endometriosis, neither the extent of their participation nor the underlying mechanisms are clearly understood. Heterogeneous uterine cell types differentially respond to estrogen and progesterone (P(4)). P(4), primarily acting via its nuclear receptor (PR), activates gene transcription and impacts many reproductive processes. Deletion of Fkbp52, an immunophilin cochaperone for PR, results in uterine-specific P(4) resistance in mice, creating an opportunity to study the unique aspects of P(4) signaling in endometriosis. Here we explored the roles of FKBP52 in this disease using Fkbp52(-/-) mice. We found that the loss of FKBP52 encourages the growth of endometriotic lesions with increased inflammation, cell proliferation, and angiogenesis. We also found remarkable down-regulation of FKBP52 in cases of human endometriosis. Our results provide the first evidence corroborated by genetic studies in mice for a potential role of an immunophilin cochaperone in the etiology of human endometriosis. This investigation is highly relevant for clinical application, particularly because P(4) resistance is favorably indicated in endometriosis and other gynecological diseases.
Abstract: Meiosis is coupled to gamete development and must be well regulated to prevent aneuploidy. During meiotic maturation, Drosophila oocytes progress from prophase I to metaphase I. The molecular factors controlling meiotic maturation timing, however, are poorly understood. We show that Drosophila alpha-endosulfine (endos) plays a key role in this process. endos mutant oocytes have a prolonged prophase I and fail to progress to metaphase I. This phenotype is similar to that of mutants of cdc2 (synonymous with cdk1) and of twine, the meiotic homolog of cdc25, which is required for Cdk1 activation. We found that Twine and Polo kinase levels are reduced in endos mutants, and identified Early girl (Elgi), a predicted E3 ubiquitin ligase, as a strong Endos-binding protein. In elgi mutant oocytes, the transition into metaphase I occurs prematurely, but Polo and Twine levels are unaffected. These results suggest that Endos controls meiotic maturation by regulating Twine and Polo levels, and, independently, by antagonizing Elgi. Finally, germline-specific expression of the human alpha-endosulfine ENSA rescues the endos mutant meiotic defects and infertility, and alpha-endosulfine is expressed in mouse oocytes, suggesting potential conservation of its meiotic function.
Abstract: Etiology of endometrial cancer (EMC) is not fully understood. Animal models with rapidly and spontaneously developing EMC will help explore mechanisms of cancer initiation and progression. Pten(+/-) mice are currently being used as a model to study EMC. These females develop atypical endometrial hyperplasia of which approximately 20% progresses to EMC. In addition, tumors develop in other organs, complicating the use of this model to specifically study EMC. Here, we show that conditional deletion of endometrial Pten results in EMC in all female mice as early as age 1 month with myometrial invasion occurring by 3 months. In contrast, conditional deletion of endometrial p53 had no phenotype within this time frame. Whereas mice with endometrial Pten deletion had a life span of approximately 5 months, mice with combined deletion of endometrial Pten and p53 had a shorter life span with an exacerbated disease state. Such rapid development of EMC from homozygous loss of endometrial Pten suggests that this organ is very sensitive to this tumor suppressor gene for tumor development. All lesions at early stages exhibited elevated Cox-2 and phospho-Akt levels, hallmarks of solid tumors. More interestingly, levels of two microRNAs miR-199a(*) and miR-101a that posttranscriptionally inhibit Cox-2 expression were down-regulated in tumors in parallel with Cox-2 up-regulation. This mouse model in which the loxP-Cre system has been used to delete endometrial Pten and/or p53 allows us to study in detail the initiation and progression of EMC. These mouse models have the added advantage because they mimic several features of human EMC.
Abstract: FKBP52 is a member of the FK506-binding family of immunophilins and serves as a co-chaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Male mice missing Fkbp52 are infertile, and this infertility has been ascribed to compromised sensitivity of the anterior prostate, external genitalia, and other accessory sex organs to androgen. Here, we show additional defects contributing to infertility. We found that epididymal Fkbp52(-/-) sperm are sparse often with aberrant morphology, and they have reduced fertilizing capacity. This phenotype, initially observed in null males on a C57BL/6/129 background, is also maintained on a CD1 background. Expression studies show that while FKBP52 and androgen receptor are co-expressed in similar cell types in the epididymis, FKBP52 is also present in epididymal sperm flagella. Collectively, our results suggest that reduced number and abnormal morphology contribute to compromised fertilizing capacity of Fkbp52(-/-) sperm. This study is clinically relevant because unraveling the role of immunophilin signaling in male fertility will help identify new targets for male contraceptives and/or alleviate male infertility.
Abstract: Reduced litter sizes in mice missing pentraxin 3 (Ptx3) have been attributed to fertilization failure. However, our global gene expression studies showed high uterine Ptx3 expression at the implantation site in mice, suggesting its role in blastocyst implantation. We initiated molecular and genetic studies in mice to explore the importance of uterine Ptx3 in this process. We found that Ptx3 is expressed in a unique and transient fashion at implantation sites. With the initiation of implantation on midnight of Day 4 of pregnancy, Ptx3 is expressed exclusively in stromal cells at the site of blastocysts. On Day 5, its expression is more intense in decidualizing stromal cells, but it disappears on Day 6. The expression again becomes evident in the deciduum on Day 7, followed by a more robust expression on Day 8, particularly at the antimesometrial pole. From Day 9, with the initiation of placentation, Ptx3 expression becomes undetectable. These results suggest a role for PTX3 in implantation and decidualization. Indeed, deletion of Ptx3 results in both compromised implantation and decidualization. Interleukin 1B (IL1B), a known inducer of Ptx3, is also transiently expressed in stromal cells at the implantation site, suggesting that IL1B is an inducer of uterine Ptx3 expression. In fact, uterine Ptx3 expression follows that of Il1b induced by lipopolysaccharide treatment on Day 7 of pregnancy. Collectively, these findings provide evidence for an important role for PTX3 in implantation and decidualization. This study has clinical implications, since PTX3 is expressed in the receptive endometrium, and trophoblast cells influence decidual Ptx3 expression in humans.
Abstract: Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.
Abstract: An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation.
Abstract: The underlying causes of epithelial ovarian cancer (EOC) are unclear, and treatment options for patients with advanced disease are limited. There is evidence that the use of nonsteroidal anti-inflammatory drugs is associated with decreased risk of developing EOC. Nonsteroidal anti-inflammatory drugs inhibit cyclooxygenase (COX)-1 and COX-2, which catalyze prostaglandin biosynthesis. We previously showed that mouse and human EOCs have increased levels of COX-1, but not COX-2, and a COX-1-selective inhibitor, SC-560, attenuates prostaglandin production and tumor growth. However, the downstream targets of COX-1 signaling in EOC are not yet known. To address this question, we evaluated peroxisome proliferator-activated receptor delta (PPARdelta) expression and function in EOC. We found that EOC cells express high levels of PPARdelta, and neutralizing PPARdelta function reduces tumor growth in vivo. More interestingly, aspirin, a nonsteroidal anti-inflammatory drug that preferentially inhibits COX-1, compromises PPARdelta function and cell growth by inhibiting extracellular signal-regulated kinases 1/2, members of the mitogen-activated protein kinase family. Our study, for the first time, shows that whereas PPARdelta can be a target of COX-1, extracellular signal-regulated kinase is a potential target of PPARdelta. The ability of aspirin to inhibit EOC growth in vivo is an exciting finding because of its low cost, lack of cardiovascular side effects, and availability.
Abstract: The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. Because microRNAs (miRNAs) have major roles in regulating gene expression, we speculated that they participate in directing the highly regulated spatiotemporally expressed genetic network during implantation. Here, we show that two miRNAs, mmu-miR-101a and mmu-miR-199a*, are spatiotemporally expressed in the mouse uterus during implantation coincident with expression of cyclooxygenase-2, a gene critical for implantation. More interestingly, our in vitro gain- and loss-of-function experiments show that cyclooxygenase-2 expression is posttranscriptionally regulated by these two miRNAs. We report on miRNA-mediated regulation of uterine gene expression in the context of implantation. We believe that many other critical genes related to this process are also regulated by miRNAs. Thus, elucidating the physiological roles of uterine miRNAs will help us better understand the genetic control of implantation, the gateway to a successful pregnancy.
Abstract: Immunophilin FKBP52 serves as a cochaperone to govern normal progesterone (P(4)) receptor (PR) function. Using Fkbp52(-/-) mice, we show intriguing aspects of uterine P(4)/PR signaling during pregnancy. Implantation failure is the major phenotype found in these null females, which is conserved on both C57BL6/129 and CD1 backgrounds. However, P(4) supplementation rescued implantation and subsequent decidualization in CD1, but not C57BL6/129, null females. Surprisingly, experimentally induced decidualization in the absence of blastocysts failed in Fkbp52(-/-) mice on either background even with P(4) supplementation, suggesting that embryonic signals complement uterine signaling for this event. Another interesting finding was that while P(4) at higher than normal pregnancy levels conferred PR signaling sufficient for implantation in CD1 null females, these levels were inefficient in maintaining pregnancy to full term. However, elevating P(4) levels further restored PR signaling to a level optimal for successful term pregnancy with normal litter size. Collectively, the results show that the indispensability of FKBP52 in uterine P(4)/PR signaling is a function of genetic disparity and is pregnancy stage specific. Since there is evidence for a correlation between P(4) supplementation and reduced risks of P(4)-resistant recurrent miscarriages and remission of endometriosis, these findings have clinical implications for genetically diverse populations of women.
Abstract: There is growing appreciation that resident glial cells can initiate and/or regulate inflammation following trauma or infection in the central nervous system (CNS). We have previously demonstrated the ability of microglia and astrocytes, resident glial cells of the CNS, to respond to bacterial pathogens by rapid production of inflammatory mediators. However, inflammation within the brain parenchyma is notably absent during some chronic bacterial infections in humans and nonhuman primates. In the present study, we demonstrate the ability of the immunosuppressive cytokine, interleukin-10 (IL-10), to inhibit inflammatory immune responses of primary microglia and astrocytes to B. burgdorferi and N. meningitidis, two disparate gram negative bacterial species that can cross the blood-brain barrier in humans. Importantly, we demonstrate that these organisms induce the delayed production of significant quantities of IL-10 by both microglia and astrocytes. Furthermore, we demonstrate that such production occurs independent of the actions of bacterial lipopolysaccharide and is secondary to the autocrine or paracrine actions of other glia-derived soluble mediators. The late onset of IL-10 production by resident glia following activation, the previously documented expression of specific receptors for this cytokine on microglia and astrocytes, and the ability of IL-10 to inhibit bacterially induced immune responses by these cells, suggest a mechanism by which resident glial cells can limit potentially damaging inflammation within the CNS in response to invading pathogens, and could explain the suppression of inflammation seen within the brain parenchyma during chronic bacterial infections.
Abstract: COX [cyclo-oxygenase; PG (prostaglandin) G/H synthase] oxygenates AA (arachidonic acid) and 2-AG (2-arachidonylglycerol) to endoperoxides that are converted into PGs and PG-Gs (glycerylprostaglandins) respectively. In vitro, 2-AG is a selective substrate for COX-2, but in zymosan-stimulated peritoneal macrophages, PG-G synthesis is not sensitive to selective COX-2 inhibition. This suggests that COX-1 oxygenates 2-AG, so studies were carried out to identify enzymes involved in zymosan-dependent PG-G and PG synthesis. When macrophages from COX-1-/- or COX-2-/- mice were treated with zymosan, 20-25% and 10-15% of the PG and PG-G synthesis observed in wild-type cells respectively was COX-2 dependent. When exogenous AA and 2-AG were supplied to COX-2-/- macrophages, PG and PG-G synthesis was reduced as compared with wild-type cells. In contrast, when exogenous substrates were provided to COX-1-/- macrophages, PG-G but not PG synthesis was reduced. Product synthesis also was evaluated in macrophages from cPLA(2alpha) (cytosolic phospholipase A2alpha)-/- mice, in which zymosan-induced PG synthesis was markedly reduced, and PG-G synthesis was increased approx. 2-fold. These studies confirm that peritoneal macrophages synthesize PG-Gs in response to zymosan, but that this process is primarily COX-1-dependent, as is the synthesis of PGs. They also indicate that the 2-AG and AA used for PG-G and PG synthesis respectively are derived from independent pathways.
Abstract: Cyclooxygenases-1 and -2 (Cox-1 and Cox-2) are two distinct isoforms that catalyze the conversion of arachidonic acid to prostaglandins. The role of Cox-2 in a variety of cancers is well recognized, but the contribution of Cox-1 remains much less explored. We have previously shown that human epithelial ovarian tumors have increased levels of Cox-1, but not Cox-2. We also observed that Cox-1 is highly expressed in a mouse model of epithelial ovarian cancer (EOC), which lacks p53 but overexpresses c-myc and K-ras or c-myc and Akt. More importantly, a Cox-1-selective inhibitor, SC-560, attenuates EOC growth. In the present investigation, we used various genetically engineered mouse models of EOC to determine whether Cox-1 overexpression is unique to specific genetic and oncogenic alterations or is widespread. These models include: (a) deletion of both p53 and Rb, (b) induction of the transforming region of SV40 under the control of Mullerian inhibitory substance type II receptor, or (c) activation of K-Ras in the absence of Pten locally in the ovarian surface epithelium. We found that these three models, which produce spontaneous EOC, also show up-regulated expression of Cox-1, but not Cox-2. The results provide further evidence that Cox-1 overexpression is common in various models of EOC. Thus, Cox-1 serves as a potential marker of EOC and is a possible target for the prevention and/or treatment of this deadly disease.
Abstract: Embryo implantation is absolutely dependent on the preparation of the uterus to the receptive stage and attainment by the blastocyst of implantation competency. Co-ordinated effects of progesterone and oestrogen are essential for these processes and determine the window of implantation. In rodents, a generalized stromal edema occurs before blastocyst attachment followed by uterine luminal closure. This leads to apposition of the blastocyst trophectoderm against the luminal epithelium and ultimately attachment. Progesterone is essential for luminal closure, which must occur for successful implantation. More importantly, progesterone is critical for almost every stage of pregnancy, including ovulation, fertilization, implantation, decidualization and pregnancy maintenance. Progesterone exerts its effects on target tissues primarily via nuclear progesterone receptor (PR) whose optimal activity is potentiated by an immunophilin co-chaperone, FK-506 binding protein 4 (FKBP52). While mice lacking PR are infertile due to complete failure of ovulation, fertilization, and implantation, female mice with targeted deletion of the Fkbp52 gene are infertile specifically because of implantation failure resulting from compromised uterine receptivity. This review highlights the evolution of knowledge about progesterone signalling during early pregnancy. Future studies are likely to provide a better understanding of FKBP52-PR signalling in promoting uterine receptivity for implantation and may reveal new targets for improving infertility.
Abstract: The process of implantation absolutely requires synchronized development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state, and a reciprocal dialogue between the blastocyst and uterine luminal epithelium. Genetic and molecular approaches have identified several signaling pathways that are critical to this process. The transcription factor Hoxa10 is one such critical player in implantation. Hoxa10-/- female mice have implantation and decidualization failure due specifically to reduced uterine responsiveness to progesterone and defective stromal cell proliferation during uterine receptivity and implantation. However, the downstream signaling pathways of Hoxa10 in these events remain largely unknown. Using the proteomics approach of difference gel electrophoresis, we have identified an immunophilin FKBP52 (FK506 binding protein 4) as one of the Hoxa10-mediated signaling molecules in the uterus. We found that FKBP52, a cochaperone protein known to influence steroid hormone receptor functions, is down-regulated in stromal cells of Hoxa10-/- mice. More importantly, FKBP52 shows differential uterine cell-specific expression during the periimplantation period. Whereas it is primarily expressed in the uterine epithelium on d 1 of pregnancy, the expression expands to the stroma on d 4 during the period of uterine receptivity and becomes localized to decidualizing stromal cells surrounding the implantation site on d 5. This suggests that FKBP52 is important for the attainment of uterine receptivity and implantation. Furthermore, FKBP52 shows differential cell-specific expression in the uterus in response to progesterone and/or estrogen consistent with its expression patterns during the periimplantation period. Collectively, these results and the female infertility phenotype of FKBP52 suggest that a Hoxa10-FKBP52 signaling axis is critical to uterine receptivity and implantation.
Abstract: Embryo implantation in the uterus is a critical step in mammalian reproduction, requiring preparation of the uterus receptive to blastocyst implantation. Uterine receptivity, also known as the window of implantation, lasts for a limited period, and it is during this period blastocysts normally implant. Ovarian steroid hormones estrogen and progesterone (P(4)) are the primary regulators of this process. The immunophilin FKBP52 serves as a cochaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Here we show a critical role for FKBP52 in mouse implantation. This immunophilin has unique spatiotemporal expression in the uterus during implantation, and females missing the Fkbp52 gene have complete implantation failure due to lack of attainment of uterine receptivity. The overlapping uterine expression of FKBP52 with nuclear progesterone receptor (PR) in wild-type mice together with reduced P(4) binding to PR, attenuated PR transcriptional activity and down-regulation of several P(4)-regulated genes in uteri of Fkbp52(-/-) mice, establishes this cochaperone as a critical regulator of uterine P(4) function. Interestingly, ovulation, another P(4)-mediated event, remains normal. Collectively, the present investigation provides evidence for an in vivo role for this cochaperone in regulating tissue-specific hormone action and its critical role in uterine receptivity for implantation.
Abstract: The precise genetic and molecular defects underlying epithelial ovarian cancer (EOC) remain largely unknown, and treatment options for patients with advanced disease are limited. Cyclooxygenases (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandins. Whereas overwhelming evidence suggests a role for COX-2 in a variety of cancers, the contribution of COX-1 remains much less explored. The expression status of COX isoforms in ovarian cancers also remains confusing. We have previously shown that human epithelial ovarian tumors have increased levels of COX-1 but not COX-2. To more carefully examine the role of COXs in ovarian cancer, we used a mouse model of EOC in which genetic and oncogenic modifications were experimentally engineered into ovarian surface epithelial cells (OSE) thought to be the cells of origin for human EOC. These OSE cells produce tumors when allografted into host mice. Using multiple approaches, we observed that OSE cells and the tumors comprised of these cells express high levels of COX-1 but not COX-2. Prostacyclin (PGI(2)) is the major prostaglandin generated downstream of COX-1 in these cells, and SC-560, a COX-1-selective inhibitor, dramatically inhibits PGI(2) production. More importantly, SC-560 reduced the growth of tumors when OSE cells were allografted in nude female mice. In contrast, the COX-2-selective inhibitor celecoxib had little effect on tumor growth. The growth inhibitory effects of SC-560 result from reduced cell proliferation and/or accelerated apoptosis. Our results imply COX-1 as a target for the prevention and/or treatment of EOC.
Abstract: Implantation is the process by which the blastocyst comes into intimate physical and physiological contact with the uterine endometrium. This process is governed by an intimate cross-talk between the activated blastocyst and the receptive uterus. An increased understanding of mammalian implantation has been gained through the use of the mouse model. This review highlights the more recently defined signaling cascades involved in this dialogue, focusing specifically on cyclooxygenase-2-derived prostaglandins, endocannabinoids, Wnt proteins, homeotic transcription factors, and immunophilins. Unraveling the nature of these signals and discovering additional molecular cascades may lead to strategies to correct implantation failure and improve pregnancy rates in women.
Abstract: Although ovarian estrogen, estradiol-17beta, is a key modulator of normal reproductive functions, natural and synthetic compounds with estrogen-like activities can further influence reproductive functions. Plant-derived phytoestrogens specifically have received much attention because of associated health benefits. However, a comprehensive understanding of the beneficial and/or detrimental impacts of phytoestrogen consumption through commercial rodent diets on uterine biology and early pregnancy at the molecular level remains largely unexplored. Using multiple approaches, we demonstrate here that exposure of adult female mice to a commercial rodent diet with higher phytoestrogen levels facilitates uterine growth in the presence or absence of ovarian estrogen, alters uterine expression of estrogen-responsive genes, and advances the timing of implantation compared with a diet with lower phytoestrogen levels. The finding that variability in phytoestrogen content in commercial rodent diets, both within and between brands, influences experimental results stresses the importance of this investigation and raises caution for investigators using rodents as animal models.
Abstract: Osteoblasts produce an array of immune molecules following bacterial challenge that could recruit leukocytes to sites of infection and promote inflammation during bone diseases, such as osteomyelitis. Recent studies from our laboratory have shed light on the mechanisms by which this cell type can perceive and respond to bacteria by demonstrating the functional expression of members of the Toll-like family of cell surface pattern recognition receptors by osteoblasts. However, we have shown that bacterial components fail to elicit immune responses comparable with those seen following challenge with the intracellular pathogens salmonellae and Staphylococcus aureus. In the present study, we show that UV-killed bacteria and invasion-defective bacterial strains elicit significantly less inflammatory cytokine production than their viable wild-type counterparts. Importantly, we demonstrate that murine osteoblasts express the novel intracellular pattern recognition receptors Nod1 and Nod2. Levels of mRNA encoding Nod molecules and protein expression are significantly and differentially increased from low basal levels following exposure to these disparate bacterial pathogens. In addition, we have shown that osteoblasts express Rip2 kinase, a critical downstream effector molecule for Nod signaling. Furthermore, to begin to establish the functional nature of Nod expression, we show that a specific ligand for Nod proteins can significantly augment immune molecule production by osteoblasts exposed to either UV-inactivated bacteria or bacterial lipopolysaccharide. As such, the presence of Nod proteins in osteoblasts could represent an important mechanism by which this cell type responds to intracellular bacterial pathogens of bone.
Abstract: Staphylococcus aureus is the single most common cause of osteomyelitis in humans. Incidences of osteomyelitis caused by S. aureus have increased dramatically in recent years, in part due to the appearance of community-acquired antibiotic resistant strains. Therefore, understanding the pathogenesis of this organism has become imperative. Recently, we have described the surprising ability of bone-forming osteoblasts to secrete a number of important immune mediators when exposed to S. aureus in vitro. In the present study, we provide the first evidence for the in vivo production of such molecules by osteoblasts during bacterial infection of bone. These studies demonstrate the expression of the key inflammatory cytokine interleukin-6 by osteoblasts in organ cultures of neonatal mouse calvaria, and in vivo using a mouse model that closely resembles the pathology of trauma-induced staphylococcal osteomyelitis, as determined by confocal microscopic analysis. Importantly, we have established the clinical relevancy of these findings in infected human bone tissue from patients with S. aureus-associated osteomyelitis. As such, these studies demonstrate that bacterial challenge of osteoblasts during bone diseases, such as osteomyelitis, induces cells to produce inflammatory molecules that can direct appropriate host responses or contribute to progressive inflammatory damage.
Abstract: Murine osteoblasts express Toll-like receptor 5 (TLR5), and this expression is upregulated following exposure to bacteria or to the TLR5 agonist, flagellin. Importantly, flagellin activates transcriptional regulators and elicits proinflammatory cytokine production, suggesting TLR5 functionality. TLR5 may represent an important mechanism underlying the recognition of bacterial pathogens by osteoblasts during bone infections.
Abstract: It has become apparent that astrocytes may be important contributors to inflammatory immune responses within the brain in response to microbial challenges. To date, the mechanisms that underlie activation of this major glial cell type by such challenges have not been investigated. In the present study, we present evidence for members of a recently discovered family of receptors for highly conserved microbial components, the Toll-like receptors (TLRs), in isolated cultures of primary murine astrocytes. We describe the low-level constitutive expression of messenger RNA-encoding TLR2, TLR4, TLR5, and TLR9 in resting cultures of these cells. Importantly, the level of expression of messenger RNA for each of these receptors is markedly elevated following exposure to specific bacteria-derived ligands for these receptors. The functional expression of these receptor proteins is further supported by the ability of known ligands for each TLR to induce both message expression and protein secretion of the proinflammatory cytokine, interleukin-6. In addition, the recent availability of antibodies to TLR2 and TLR4 has enabled us to demonstrate directly the presence of these receptors on astrocytes by Western blot and immunofluorescence analysis, respectively. Furthermore, we have confirmed the sensitivity of such receptor expression to ligand stimulation. The present demonstration of Toll-like microbial pattern-recognition receptors on primary astrocytes provides a mechanistic link between bacterial challenge and inflammatory immune responses that may be an important component of the pathologies of bacterially induced inflammatory CNS disorders.
Abstract: Nitric oxide (NO) production plays an important role in regulating preimplantation embryo development. NO is produced from l-arginine by the enzyme nitric oxide synthase (NOS), which has three isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). It has been previously shown that inhibition of NO production by NG-nitro-l-arginine (l-NA) inhibits the development of two-cell embryos to the four-cell stage. However, excess NO also halts embryo development, possibly through the production of free radicals. We hypothesize that multiple NOS isoforms are expressed in order to ensure normal preimplantation embryo development and that, in this process, NO acts through the cGMP pathway. Using reverse transcription-polymerase chain reaction, mRNA for all three NOS isoforms was amplified from two-cell, four-cell, morula, and blastocyst embryos. However, blastocyst-stage embryos isolated midmorning on Day 4 of pregnancy expressed only nNOS and eNOS, whereas those isolated midafternoon again expressed all three NOS isoforms. Culture of one-cell embryos in various concentrations of Whitten (positive control), S-nitroso-N-acetylpenicillamine (SNP, a NO donor), l-NA, and/or 8-Br-cGMP demonstrated that NO is acting, at least in part, through cGMP in preimplantation embryo development. In addition, we determined that a critical concentration of NO and cGMP is required for normal embryo development and deviations from this concentration lead to developmental arrest and/or apoptosis of the embryo. This data provides support for a requirement of NO in preimplantation embryo development and one mechanism through which it regulates mitotic division in these embryos.
Abstract: Nitric oxide acts as an important intracellular messenger in a variety of systems, including reproduction. Previous studies have shown the importance of nitric oxide in embryo development. NO is produced from l-arginine by the enzyme, nitric oxide synthase (NOS), which has three isoforms: endothelial (NOS3), neural (NOS1), and inducible (NOS2). We hypothesize that, because of the importance of NOS in development, at least two NOS isoforms are required in order for normal embryo development to occur. Through the generation of NOS3/NOS2, NOS3/NOS1, and NOS2/NOS1 double knockout mice, we found that while litter size remains unchanged, the expected number of generated double knockout mice varies significantly from what would be predicted by Mendelian genetics. Estrous cycles were similar for both DKO and the wild-type mice, and both groups were deemed fertile by their ability to mate with wild-type (CD-1) mice. Together, these results lead us to conclude that the lack of two NOS isoforms leads to a decreased viability in mice because of a developmental problem in the double knockout embryo.
