GATA6 expression promoted by an active enhancer may become a molecular marker in endometriosis lesions.

PROBLEM: Genome-wide profiling of DNA methylation in endometriotic cells has shown a distinct facet of epigenetic backgrounds; however, specific DNA methylation sites responsible for aberrant gene expression in endometriosis were unknown. Are there specific endometriosis-associated DNA methylations that can be used as molecular markers in endometriosis lesions? METHOD OF STUDY: This study used endometriotic tissues from the chocolate cyst lining of the ovaries of patients with endometriosis, and endometrial tissues from disease-free patients. For analysis, stromal cells were collected from endometrial and endometriotic tissues. Using endometrial cells as control, differentially methylated cytosine-phosphate-guanine (CpG) characteristic in endometriotic cells was extracted. Among these CpGs, we focused on a stretch of hypomethylated CpGs within GATA6 gene and examined the potential role as enhancer in endometriotic cells and tissues. RESULT(S): We identified a stretch of hypomethylated CpGs within the GATA6 gene body in endometriotic cells. Because GATA6 mRNA was highly expressed in endometriotic cells but not in endometrial cells, we then hypothesized that the hypomethylated sequence may function as an enhancer in GATA6 gene expression. Chromatin immunoprecipitation analysis predicted the presence of active enhancer within the gene body sequence in endometriotic cells. Immunohistochemistry showed a positive staining of GATA6 in ovarian chocolate cysts, while in endometrial tissues and in some peritoneal tissues with endometriosis, GATA6 staining was at a marginal level. CONCLUSION: This is the first implication showing a link between an aberrant DNA methylation of cis element and gene expression in endometriosis. GATA6 expression may become a molecular marker to diagnose endometriosis lesions.

Lipopolysaccharide promotes the development of murine endometriosis-like lesions via the nuclear factor-kappa B pathway.

PROBLEM: Is lipopolysaccharide (LPS) involved in the development of endometriosis? METHOD OF STUDY: BALB/c mice (n=69) were used for the murine endometriosis model. Mice with surgically induced endometriosis were injected with LPS intraperitoneally. After 4 weeks of LPS injections with or without the nuclear factor-kappa B (NF-κB) inhibitor, the extent of endometriosis-like lesions was evaluated. Expression of inflammatory factors in the implants was evaluated using real-time RT-PCR. Cell proliferation, angiogenic activity, inflammation, and NF-κB phosphorylation were assessed by immunohistochemical staining. RESULTS: Lipopolysaccharide increased total number, size, and mRNA expression of Ptgs-2, Vegf, Ccl-2, and Il-6 in endometriosis-like lesions. LPS also increased the percentage of Ki67-positive cells and enhanced the intensity and rate of positive cells of CD3, F4/80, and PECAM. Intense expression of phospho-NF-κB p65 after LPS administration was observed. Treatment with the NF-kB inhibitor negated these LPS-induced effects. CONCLUSION: LPS-induced pelvic inflammation status enhanced the development of murine endometriosis-like lesions via NF-κB pathway.

Molecular Background of Estrogen Receptor Gene Expression in Endometriotic Cells.

The molecular background of estrogen receptor (ER) expression is important to understand the pathophysiology of the high estrogen environment in endometriosis. However, the molecular details have not been fully understood. The objective of this study is to evaluate the molecular background of ERα and ERß messenger RNA (mRNA) expression in endometriotic cells. The following summarizes our observations: (1) ERα mRNA expression in endometriotic cells was estimated to be approximately one-tenth of that in endometrial cells. (2) Three mRNAs, which include 3 different 5'-untranslated exons tagged to an open reading frame of wild-type ERα, were detected. (3) Expression of ERß mRNA depends mostly on 0N promoter and includes 2 open reading frames: one for a wild-type ERß1 and another for a splice variant ERß2. (4) Expression of ERß1 mRNA was approximately 40-fold higher than that in endometrial cells. (5) Expression of ERß2 mRNA was almost at a comparable level of the ERß1. 9 (6) ERα and ERß mRNAs are equivalently expressed in endometriotic cells. These observations show the molecular background of ER mRNA expression in endometriotic cells and provide a clue to further understanding the estrogen-dependent pathophysiology leading to clinical application in endometriosis.

The cellular inhibitor of apoptosis protein-2 is a possible target of novel treatment for endometriosis.

PROBLEM: How is the tumor necrosis factor (TNF) α-induced inhibitor of apoptosis (IAP) protein expression in endometriotic stromal cells (ESCs) involved in cell viability and signaling pathways? METHOD OF STUDY: Endometriotic stromal cells were isolated from ovarian chocolate cysts in 20 patients who underwent laparoscopic surgery. IAP protein expression and IκB phosphorylation were evaluated by Western blot analysis. Interleukin (IL)-8 protein expression and cell proliferation were assessed by ELISA. RESULTS: Cellular IAP (cIAP)-2 protein expression in endometriotic tissue was higher than that of endometrium. TNFα markedly enhanced cIAP-2 protein expression in ESCs. Pretreatment with a nuclear factor (NF)-κB inhibitor attenuated TNFα-induced cIAP-2 expression. An antagonist of IAPs abrogated TNFα-induced cIAP-2 protein expression and showed a decrease in TNFα-induced IL-8 protein expression and BrdU incorporation in ESCs. CONCLUSIONS: TNFα and its downstream NFκB pathway have proven to be critical regulators of highly expressed cIAP-2 in ESCs. cIAP-2 may be a novel therapeutic target for endometriosis.

Epigenetic aberration of gene expression in endometriosis.

Endometriosis is an estrogen-dependent inflammatory disease. In endometriotic tissues, a high-estrogen environment associated with up-regulation of the aromatase gene has been well documented. There is accumulating evidence supporting a concept that endometriosis is a disease associated with an epigenetic disorder. Epigenetics is one of the most expanding fields in the current biomedical research. The word 'epigenetics' refers to the study of mitotically and/or meiotically heritable changes in gene expression that occur without changes in the DNA sequence. The disruption of such changes (epigenetic aberration or disorder) underlies a wide variety of pathologies. Epigenetic regulation includes DNA methylation and histone modifications, and is responsible for a number of gene transcription associated with chromatin modifications that distinguish the states of diseases. In this review, we summarized our studies as well as recent studies from other laboratories using an epigenetic approach focused on DNA methylation. We also summarized studies using advanced technologies including Genome-Wide (GW) methylation profiling analysis and GW Association Study (GWAS). We reviewed recent monozygotic twins studies in relation to environmental factors since they may provide insight into the epigenetic background of endometriosis. Finally, we referred to a new concept of GW DNA methylation.

Apoptosis and endometriosis.

Apoptosis is a distinctive form of programmed cell death resulting in the efficient elimination of cells without eliciting an inflammatory response. Endometriosis is characterized by the presence of endometrial cells with capacity to avoid apoptosis outside the uterus. Apoptosis plays a fundamental role for the pathogenesis of endometriosis. Eutopic endometrium from women with endometriosis has increased expression of anti-apoptotic factor and decreased expression of pro-apoptotic factors compared with endometrium from healthy women. These differences could contribute to the survival of regurgitating endometrial cells into the peritoneal cavity and development of endometriosis. Increased apoptosis of Fas-bearing immune cells in the peritoneal cavity may leads to their decreased scavenger activity that eventually results in prolonged survival of ectopic endometrial cells in women with endometriosis. This study is a current review of the literatures focused on the physiological role of apoptosis in normal endometrium and alterations in regulation of apoptosis in eutopic and ectopic endometrium from women with endometriosis. The role of apoptosis in the treatment of endometriosis is also reviewed.

Demethylation of a nonpromoter cytosine-phosphate-guanine island in the aromatase gene may cause the aberrant up-regulation in endometriotic tissues.

OBJECTIVE: To search for the demethylated cytosine-phosphate-guanine (CpG) islands within the aromatase gene in stromal cells derived from endometriotic chocolate cysts. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology and Department of Biosignaling, Tottori University, Yonago, Japan. PATIENT(S): Twenty-eight women who underwent laparoscopy (n=14) and laparotomy (n=14). INTERVENTION(S): Endometrial and endometriotic stromal cells were obtained from the uterus and chocolate cyst lining of the ovary. MAIN OUTCOME MEASURE(S): We searched for the CpG island and examined methylation profile and the association of methyl-binding proteins with the CpG island. RESULT(S): Up-regulation of aromatase messenger RNA (mRNA) expression was demonstrated in endometriotic cells. Three proximal promoters drove the mRNA expression. In endometrial cells, a marginal level of aromatase mRNA expression was observed. Treating endometrial cells with the demethylating agent 5-aza-2'-deoxycytidine markedly enhanced aromatase mRNA expression. The same promoters as in the endometriotic cells were used. To identify the unmethylated CpGs in endometriotic cells, we searched for CpG islands within the aromatase gene and subsequently examined the methylation profiles. Sequence analysis of bisulfite-treated genomic DNA demonstrated a stretch of CpG demethylation within a nonpromoter CpG island of the aromatase gene in endometriotic cells. In endometrial cells, the CpG sequences were heavily methylated and associated with methyl-CpG-binding proteins. CONCLUSION(S): The up-regulation of the aromatase gene in endometriosis may be ascribed to the epigenetic disorder associated with aberrant DNA demethylation in a nonpromoter CpG island.

The role of survivin in the resistance of endometriotic stromal cells to drug-induced apoptosis.

BACKGROUND: Decreased susceptibility of endometrial tissue to apoptosis may contribute to the pathogenesis of endometriosis. We investigate the role of survivin in the pathophysiology of endometriosis through the ability of ectopic and eutopic endometrial stromal cells (ESCs) to resist apoptosis. METHODS: Ectopic ESCs were obtained from ovarian chocolate cysts in patients undergoing laparoscopic surgery (n = 22). Eutopic ESCs were isolated from endometrial tissue of cyclic premenopausal women undergoing hysterectomy for fibroids (n = 22). Purified stromal cells were studied in vitro. The number of surviving cells and activation of caspases were assessed by WST-8 assay and immunoblotting. Expression of inhibitor of apoptosis proteins (IAP) family members: cIAP1 (birc2), cIAP2 (birc3), XIAP (birc4), survivin (birc5) were examined using cDNA array and real-time RT-PCR. Effects of gene silencing by small inhibitor RNAs (siRNA) were examined by WST-8-assay, Annexin-V staining and immunoblotting. RESULTS: After staurosporine (SS) treatment, 55% of eutopic ESCs survived versus 70% of ectopic ESCs. Procaspase-3 or -7 was more intensely activated by SS treatment in eutopic than in ectopic ESCs (P < 0.01). mRNAs for IAP-family genes, such as cIAP-1, XIAP and survivin, were highly expressed in ectopic ESCs before SS treatment. The fold induction of survivin expression after SS treatment was higher in ectopic than eutopic ESCs (2.8 +/- 0.27 versus 0.69 +/- 0.07, respectively). Survivin gene silencing in SS-treated ectopic ESCs led to an increase of apoptotic cells (P < 0.05, versus control siRNA). CONCLUSIONS: We demonstrated that survivin plays a critical role in susceptibility of ESCs to apoptosis. Our results indicate that a survivin inhibitor may be effective as a novel treatment for endometriosis.

Cytochrome P450 aromatase gene (CYP19) expression in gastric cancer.

BACKGROUND: The secretion of biologically active estrogen through the conversion of circulating precursor androgens has been suggested to play important roles in the pathophysiology of estrogen-dependent carcinomas. In the present study, we examined aromatase expression in gastric carcinoma. METHODS: Nineteen specimens of gastric carcinoma were obtained from Japanese patients at the Department of Surgery, Tottori University Hospital, Japan. Nontumoral tissues adjacent to the carcinoma were also available for analysis. The histological features of the gastric carcinomas were as follows: 8 intestinal-type and 11 diffuse-type adenocarcinomas. Tissue specimens removed at surgery were used for the preparation of RNA or for immunohistochemical analysis. Six cell lines derived from human gastric cancers were also used as a model system. Using conventional and real-time reverse transcription-polymerase chain reactions, aromatase mRNA expression and promoter usage were assayed. Immunohistochemical analysis was performed using an anti-aromatase antibody. RESULTS: We demonstrated the molecular basis of aromatase mRNA expression, which depended on three proximal promoters in tumoral and nontumoral tissues, for the first time. The tumoral tissues exhibited positive staining for anti-aromatase antibody. At the same time, positive staining was also observed in nontumoral mucosa, predominantly in the parietal cells. CONCLUSION: We provide evidence suggesting a mechanism for the secretion of estrogen through the conversion of a precursor androgen in tumoral and nontumoral tissues in the stomach.

An epigenetic disorder may cause aberrant expression of aromatase gene in endometriotic stromal cells.

OBJECTIVE: To examine the molecular basis of aromatase expression in stromal cell culture from endometriotic chocolate cysts. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology and Department of Biosignaling, Tottori University, Yonago Japan. PATIENT(S): Thirty women, selected randomly, who underwent laparoscopy (n = 18) or laparotomy (n = 12). INTERVENTION(S): Endometrial and endometriotic stromal cells were obtained from the uterus and chocolate cyst lining of the ovary. MAIN OUTCOME MEASURE(S): Estradiol concentrations in the culture media were measured by means of enzyme immunoassay. Aromatase expression was examined by quantitative real-time polymerase chain reaction. Promoter usage was examined using unique exon I (PII, I.1, I.3, I.4, I.5, and I.6) and exon II primers. To determine the effect of 5-aza-deoxycytidine on endometrial stromal cells, the cells were treated with the agent for 96 hours. RESULT(S): Endometriotic cells secreted a marginal level of estradiol into the culture media, but adding testosterone to the culture produced a pronounced level of estradiol. In endometrial cells, estradiol production was far less efficient than in endometriotic cells even after adding testosterone. Real-time polymerase chain reaction analyses demonstrated the up-regulation of aromatase messenger RNA (mRNA) expression in endometriotic cells. Three proximal promoters, PII, 1.3, and 1.6, drove mRNA expression. In endometrial cells where a marginal level of aromatase mRNA expression was observed, the same promoters as those in the endometriotic cells were used. To determine the role of epigenetic modification of aromatase gene expression in endometriotic cells, endometrial cells were treated with 5-aza-deoxycytidine, which markedly enhanced aromatase mRNA expression, depending on the same proximal promoters as those in endometriotic cells. CONCLUSION(S): An epigenetic disorder may play a role in the pathophysiology of endometriosis.

Apoptosis and endometriosis.

Apoptosis plays a critical role in maintaining tissue homeostasis and represents a normal function to eliminate excess or dysfunctional cells. Accumulated evidence suggest that apoptosis helps to maintain cellular homeostasis during the menstrual cycle by eliminating senescent cells from the functional layer of the uterine endometrium during the late secretory and menstrual phase of the cycle. BCL-2 family and Fas/FasL system have been extensively studied in human endometrium and endometriotic tissues. Eutopic endometrium from women with endometriosis reportedly has some fundamental differences compared with normal endometrium of women without endometriosis. The differences could contribute to the survival of regurgitating endometrial cells into the peritoneal cavity and the development of endometriosis. One mechanism that recently gained a lot of interest is the finding that apoptosis appeared in eutopic and ectopic endometrium of patients with endometriosis. This study is a current review of the literature focused on the physiological role of apoptosis in normal endometrium and the alterations in regulation of apoptosis in eutopic and ectopic endometrium from women with endometriosis. Finally, role of apoptosis in the treatment of endometriosis is reviewed to link the basic research findings into clinical applications.

Anaphylactic shock caused by exposure to sea anemones.

BACKGROUND: Since the first report of a dog that developed severe systemic symptoms in response to a second injection of sea anemone toxin by Richet and Portier in 1902, no clear human cases of anaphylaxis related to exposure to sea anemones has been reported in the literature. METHODS: A 24-year-old man with an episode of local urticaria on his first contact with a sea anemone (Stichodactyla haddoni), developed dyspnea, severe urticaria and hypotension on exposure to water containing the dead bodies of the organism. To study whether this reaction was mediated by antigen-specific IgE, we performed a histamine release test with blood, Western blotting with serum and lymphocyte proliferating test with peripheral blood mononuclear cells of the patient, for the homogenate of sea anemones. RESULTS: The homogenate of sea anemones induced histamine release from the blood of the patient, but it also induced histamine release from the blood of control subjects. Moreover, it also caused hemolysis of blood of all donors. However, Western-blotting demonstrated the presence of an 86 kd protein-specific IgE in the serum of the patient. CONCLUSIONS: Protein antigen(s) in sea anemones may cause anaphylactic shock under the influence of the cytolytic effects and/or lymphocyte-stimulating activity elicited by the toxin of sea anemones.

Drug-induced apoptosis was markedly attenuated in endometriotic stromal cells.

BACKGROUND: The survival of endometriotic cells in the ectopic site has been investigated from the aspect of susceptibility of endometriotic tissues to apoptosis. In order to investigate the nature of abnormal survival of endometriotic cells in ectopic locations, we compared drug-induced apoptosis in endometrial and endometriotic cells. METHODS: Endometrial stromal cells were obtained from normal endometrium in 11 patients who underwent hysterectomy for leiomyoma without endometriosis. Endometriotic cells were isolated from the chocolate cyst linings of the ovary in 13 patients who underwent laparoscopic surgery. Cells were cultured in the presence or absence of staurosporine. Apoptotic cell death was evaluated by staining nuclei with propidium iodide and phosphatidylserine (a marker of early apoptotic events) with Annexin V as well as by DNA fragmentation assay. The number of viable cells was estimated by modified MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide WST-8] assay. RESULTS: After 3 h of exposure to staurosporine, >50% of the endometrial stromal cells became Annexin V positive. In contrast, >30% of the endometriotic cells were Annexin V positive. DNA fragmentation was not clearly induced in the endometriotic cells. Less than 20% of the endometrial cells survived after staurosporine exposure, while >40% of the endometriotic cells survived. Cell death induced by staurosporine was partially blocked by incubation with the caspase inhibitor, N-benzyoxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl-ketone (ZVAD-fmk), suggesting that a caspase cascade may play a role in the cell death process. CONCLUSIONS: Attenuated susceptibility to apoptosis in endometriotic stromal cells may be associated with abnormal survival in ectopic sites in an environment that is probably unfavourable. These results may be implicated in the pathophysiology of endometriosis.

Reduction of estrogen production by interleukin-6 in a human granulosa tumor cell line may have implications for endometriosis-associated infertility.

OBJECTIVE: To examine the effect of interleukin-6 (IL-6) on estrogen production and aromatase activity using a human granulosa tumor cell line (KGN cells). The involvement of the mitogen-activated protein kinase (MAPK) cascade in the inhibitory effects of IL-6 on estrogen production was also evaluated. DESIGN: Molecular and biological studies of KGN cells. SETTING: Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. MAIN OUTCOME MEASURE(S): Gene expression of IL-6 and the IL-6 receptor was analyzed by reverse transcription-polymerase chain reaction and Southern blot analysis. KGN cells were cultured for 48 hours with IL-6 (0.1-10 ng/mL) or IL-6 (10 ng/mL) plus a mitogen activated protein kinase-extracellular signal regulated kinase kinase 1/2 (MEK1/2) inhibitor U0126 (10 microM). Estradiol concentration in the culture supernatants was measured by means of enzyme immunoassay, [1beta-(3)H] androstenedione was added to the cell lysate supernatant, and aromatase activity was determined by measuring the amount of [(3)H] H(2)O released upon the conversion of [1beta-(3)H] androstenedione to estrone. To examine the activation of intracellular signal transduction molecules induced by IL-6, the phosphorylation of Stat3, p38 MAPK, and extracellular signal-regulated kinase 1/2 (ERK1/2) was examined by Western blotting. RESULT(S): Gene expression of IL-6 and its receptor was detected in KGN cells. Estradiol secretion was significantly inhibited by adding IL-6, which also suppressed aromatase activity to 50% of the control. In addition, pretreatment with U0126 restored the IL-6-induced suppression of aromatase activity. IL-6 markedly enhanced the phosphorylation of ERK1/2, but not Stat3 and p38 MAPK. U0126 markedly reduced the level of the IL-6-induced phosphorylation of ERK1/2. CONCLUSION(S): These findings demonstrate that IL-6 may reduce estrogen production via the MAPK signal pathway in human granulosa cells. The results may support the notion that IL-6 is related to impaired estrogen biosynthesis in patients with endometriosis.