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.

Parthenolide reduces cell proliferation and prostaglandin E2 [corrected] in human endometriotic stromal cells and inhibits development of endometriosis in the murine model.

OBJECTIVE: To evaluate the effects of parthenolide on human endometriotic cells and murine endometriotic lesions. DESIGN: Experimental study. SETTING: University hospital and laboratory of animal science. PATIENT(S) AND ANIMAL(S): Twenty women with ovarian endometrioma and 30 mice. INTERVENTION(S): Ectopic endometrial tissue from the endometrioma was collected. MAIN OUTCOME MEASURE(S): Human endometriotic stromal cells (ESCs) were pretreated with parthenolide and exposed to tumor necrosis factor (TNF)-α. Interleukin 8 (IL-8) and COX-2 gene expressions were evaluated by real-time reverse transcription-polymerase chain reaction. Interleukin-8 protein, prostaglandin E2 (PGE2) level, and intranuclear p65 protein concentration were determined by ELISA. Cell proliferation was assessed by 5-bromo-2'-deoxyuridine-ELISA. Phosphorylation of signaling pathways in ESCs was evaluated by Western blotting. Gene expression and proliferative activity in murine endometriosis-like lesions were assessed by real-time reverse transcription-polymerase chain reaction and Ki67 staining, respectively. RESULT(S): With parthenolide pretreatment, TNF-α-induced IL-8 gene and protein expression in ESCs were diminished. Tumor necrosis factor α-induced COX-2 expression and PGE2 synthesis were also inhibited. Adding parthenolide repressed TNF-α-induced 5-bromo-2'-deoxyuridine incorporation and IκB phosphorylation in ESCs. As in vivo experiments, administering parthenolide reduced the number, surface area, and weight, the level of Vegf, Il-6, Mcp-1, and Lif gene expression, and the percentage of Ki67-positive cells in murine endometriosis-like lesions. CONCLUSION(S): Parthenolide repressed the development of endometriosis by suppressing the inflammatory peritoneal environment through the nuclear factor κB pathway.

Gonadotropin-releasing hormone analogues reduce the proliferation of endometrial stromal cells but not endometriotic cells.

AIMS: We investigated the potential of gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists to inhibit cell proliferation in endometriotic and endometrial stromal cells. METHODS: Twenty patients with ovarian endometriomas and 18 patients with uterine fibromas were recruited. Endometriotic and endometrial stromal cells were obtained from the ovarian chocolate cyst linings and the eutopic endometria of premenopausal women with uterine fibromas, respectively. RESULTS: GnRH agonist or antagonist treatment attenuated tumor necrosis factor (TNF)-α-induced cell proliferation in the endometrial stromal cells, whereas endometriotic stromal cells did not respond to treatment. The endometriotic stromal cells exhibited a decreased expression of the type I GnRH receptor compared with the endometrial stromal cells. GnRH agonists or antagonists did not repress TNF-α-induced IL-8 production in endometriotic stromal cells. CONCLUSION: GnRH agonists and antagonists have similar effects in slowing the growth of endometrial stromal cells. Endometriotic stromal cells resist the antiproliferative effect of GnRH agonists and antagonists.

The role of NF-kappaB in endometriosis.

The nuclear factor kappaB (NF-kappaB) is a ubiquitously expressed transcription factor playing vital roles in innate immunity and other processes involving cellular survival, proliferation, and differentiation. This review highlights the importance of NF-kappaB in the pathophysiology of endometriosis. Constitutive activation of NF-kappaB has been shown in endometriotic lesions. Complex interactions of NF-kappaB with steroid receptors and apoptotic molecules in endometriosis resulting in opposing roles of NF-kappaB are discussed. NF-kappaB regulates the expression of cytokines mediating autocrine self-amplifying cycles of cytokine release and NF-kappaB activation, leading to maintenance of inflammatory reactions in endometriosis. NF-kappaB can contribute to the increased ability of endometriotic cells to invade and adhere to the peritoneal surface by regulating the expression of matrix metaloproteinases. We are presenting the role of NF-kappaB to regulate vascularization and oxidative stress in endometriotic cells. Effects of drugs used for the treatment of endometriosis on NF-kappaB pathway are presented and we show how drugs that inhibit the NF-kappaB can mediate the progression of endometriosis. Novel therapeutic strategies involving the NF-kappaB and applied in endometriosis are also discussed.

The importance of venous Doppler velocimetry for evaluation of intrauterine growth restriction.

The management of growth-restricted fetuses requires accurate diagnosis to optimize the timing of delivery. Doppler velocimetry is the only noninvasive method for assessing the fetoplacental hemodynamic status. This review will give a critical overview of the current knowledge on fetal venous blood flow in pregnancies complicated by in-trauterine growth-restricted fetuses. Adaptation of the circulation in intrauterine growth-restricted fetuses is described. Normal and abnormal venous Doppler waveforms are presented. Correlations of abnormal waveforms with the presence of acidemia and perinatal outcomes are emphasized. Limitations of venous Doppler velocimetry for optimizing the time for delivery and the perinatal outcome are also presented.

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.

TAK1 activation for cytokine synthesis and proliferation of endometriotic cells.

Endometriosis causes pelvic pain and infertility in women of reproductive age. We explored TNFalpha-induced specific signaling pathways and gene expressions in endometriotic stromal cells (ESCs). Based on the data of the pathway specific cDNA array, we analyzed the role of TAK1, which is believed to work as a common mediator for NF-kappaB and MAPK pathways. Using the NF-kappaB pathway array, we found that TNFalpha upregulated ICAM-3, IL-6, IL-8, TAK1, JNK2, RelA, and TLR4 expressions. TNFalpha augmented the phosphorylation of TAK1. By transfection of TAK1 siRNA, TNFalpha-induced phosphorylation of IkappaBalpha, JNK1/2, and p38MAPK, as well as IL-6 or IL-8 expression, were repressed. TAK1 silencing in TNFalpha-pretreated ESCs caused a decrease in the proportion of cells in S-phase, and reduced TNFalpha-promoted BrdU incorporation. We provide the first evidence that TNFalpha and its downstream TAK1, which are key mediators for NF-kappaB and MAPK pathways, may be involved in the pathogenesis of endometriosis.

Detailed multigrade evaluation of fetal disorders with the quantified actocardiogram.

AIMS: To evaluate fetal disorders using detailed quantitative values from the actocardiogram (ACG) involving simultaneous tracing of ultrasonic Doppler fetal movement bursts and fetal heart rate (FHR). METHODS: Duration of FHR accelerations and fetal movement bursts were measured manually in 20 common fetal disorders. The severity of the fetal disorder was estimated using the FHR acceleration duration to movement burst ratio (A/B ratio) and 10-0 clinical severity ranks derived from the A/B ratio. The correlation of the A/B ratio and 1 and 5 min Apgar scores, as well as numerically expressed long-term outcomes were studied. RESULTS: A/B ratios were significantly correlated with the 1 and 5 min Apgar scores and the numerically evaluated long-term outcomes. Controversial cases of FHR pattern were more easily understood using the A/B ratio. The 10-0 severity derived from the A/B ratio was useful in clinical fetal studies. CONCLUSION: Common fetal disorders were evaluated quantitatively and in more detail using the A/B ratio from the actocardiogram than when using common binary good or bad evaluation. The A/B ratio was useful in outcome estimation, where the prognostic capability of the A/B ratio was confirmed by significant correlation with 1 and 5 min Apgar scores and long-term outcomes of fetal disorders.

Serum interleukin-8 levels are elevated in patients with ovarian endometrioma.

OBJECTIVE: To determine whether serum interleukin (IL)-8 concentration can be measured in patients with ovarian endometrioma and whether this measurement is a useful tool in diagnosing this disease. DESIGN: A controlled clinical study and an in vitro study. SETTING: Department of Obstetrics and Gynecology, Tottori University, Japan. PATIENT(S): Seventy patients with ovarian endometrioma and 21 patients with benign ovarian cyst. INTERVENTION(S): Laparoscopic surgery or laparotomy for ovarian endometriomas or benign ovarian cyst was performed. Preoperative blood samples were obtained. Endometriotic stromal cells obtained from nine patients with endometrioma were cultured. MAIN OUTCOME MEASURE(S): Interleukin-8 concentration in the serum or supernatant of the cell culture was measured with use of ELISA. RESULT(S): The serum concentration of IL-8 in patients with endometrioma was significantly higher than in patients with benign ovarian cyst. The serum IL-8 threshold (25 pg/mL) had a higher sensitivity (71.4%) for diagnosing ovarian endometrioma than did serum CA-125 level. The increased rates of IL-8 concentration in the culture supernatants after adding tumor necrosis factor alpha were significantly higher in patients whose serum IL-8 levels were >or=25 pg/mL than in those with levels <25 pg/mL. CONCLUSION(S): Measuring of serum IL-8 concentration may be a valuable tool in diagnosing endometriosis.

Peroxisome proliferator-activated receptor-gamma ligand reduced tumor necrosis factor-alpha-induced interleukin-8 production and growth in endometriotic stromal cells.

OBJECTIVE: To evaluate the influence of peroxisome proliferator-activated receptor-gamma (PPAR gamma) ligand (pioglitazone) on tumor necrosis factor-alpha (TNF-alpha)-induced interleukin-8 (IL-8) expression in endometriotic stromal cells (ESCs) and on proliferation of ESCs. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. PATIENT(S): Twenty-seven patients who underwent laparoscopic surgery. INTERVENTION(S): The ESCs were obtained from the chocolate cyst linings of ovaries. MAIN OUTCOME MEASURE(S): The expression of PPAR gamma gene and protein was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. We determined the effect of pioglitazone on the production of TNF-alpha-induced IL-8 protein in culture supernatant of ESCs using ELISA. The effect of pioglitazone on TNF-alpha-induced proliferation of ESCs was evaluated by 5-bromo-2'-deoxyuridine proliferation assay. The activation of nuclear factor (NF)-kappaB in ESCs was evaluated by Western blot analyses and NF-kappaB transcription factor assays. RESULT(S): Immunocytochemistry and RT-PCR revealed the expression of PPAR gamma gene and protein in ESCs. The PPAR gamma protein was predominantly located in the cell nucleus. Measurement of IL-8 protein by ELISA showed that adding TNF-alpha (100 pg/mL) significantly increased IL-8 protein. Treating ESCs with 0.1-10 microM of pioglitazone significantly reduced the TNF-alpha-induced IL-8 production. The presence of 0.1-10 microM of pioglitazone significantly suppressed growth of ESCs. The TNF-alpha increased the expression of phosphorylation of inhibitor kappaB (I kappaB). Adding pioglitazone (10 microM) did not influence the expression of phosphorylated inhibitor kappaB (I kappaB). The TNF-alpha markedly increased the intranuclear concentration of p65, and adding pioglitazone (10 microM) significantly reduced the concentration of p65. CONCLUSION(S): The present study demonstrates for the first time that PPAR gamma is expressed in ESCs, and that pioglitazone reduced IL-8 secretion and the proliferation of ESCs. The PPAR gamma ligand may be an attractive therapeutic agent for endometriosis.

Aberrant expression of keratinocyte growth factor receptor in ovarian surface epithelial cells of endometrioma.

Ovarian surface epithelial cells (OSEs) are considered to be the common source of endometrioma and epithelial ovarian cancer. The present study reveals that keratinocyte growth factor receptor (KGFR) messenger RNA was expressed in OSEs of endometriomas but not in those of normal ovaries, suggesting that autocrine KGF/KGFR and paracrine fibroblast growth factor 10/KGFR signaling loops may be involved with the proliferation in OSEs of endometrioma.

Involvement of p38 MAP kinase in lipopolysaccharide-induced production of pro- and anti-inflammatory cytokines and prostaglandin E(2) in human choriodecidua.

Chorioamnionitis is implicated in the pathogenesis of preterm delivery. However, the detailed mechanisms by which infection induces preterm labor are not well understood. This study has assessed the involvement of mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-induced pro- and anti-inflammatory cytokine and prostaglandin (PG) production in human choriodecidua. Samples of choriodecidua were collected before the onset of labor from women undergoing elective cesarean sections at term for breech presentation, previous cesarean delivery or cephalopelvic disproportion. Concentrations of TNFalpha, IL-10, PGE(2) and PGF(2)alpha in culture supernatants were measured by ELISA. Expression of COX-2 protein was analyzed by Western blotting. In human choriodecidual explants, LPS induced TNFalpha and IL-10 production in a dose- and time-dependent manner. LPS also up-regulated COX-2 expression and PG synthesis. Phosphorylations of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal kinases (JNK) were also confirmed by Western blotting. Furthermore, the effect of MAPK inhibitors was examined on LPS-induced pro- and anti-inflammatory cytokines and PG synthesis. Among the MAPK inhibitors examined, the p38 MAPK inhibitor, SB202190, significantly suppressed LPS-induced cytokine and PG production. SB202190 most profoundly suppressed the TNFalpha to IL-10 ratio, demonstrating that p38 MAPK inhibitor reduced predominantly TNFalpha other than IL-10 production. Phospho-p38 MAPK immunostaining was intense in extravillous trophoblast cells. The p38 MAPK seems to be most involved in signaling mechanisms when infection and inflammation cause preterm labor through PG synthesis. Novel therapeutic modalities targeting p38 MAPK may prevent to arrest preterm labor.

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.

Macrophage-activating lipopeptide-2 induces cyclooxygenase-2 and prostaglandin E(2) via toll-like receptor 2 in human placental trophoblast cells.

We have examined whether toll-like receptor (TLR)2-mediated stimulation by macrophage-activating lipopeptide-2 (MALP-2), originally purified from Mycoplasma fermentans, induces cyclooxygenase (COX)-2 and prostaglandin (PG)E(2) in human placental trophoblast cells. The signaling mechanism by which MALP-2 exerts its effect has also been examined. Human placental trophoblast cells isolated from term placenta were used. TLR expression in trophoblast cells was confirmed by multiplex PCR and immunocytochemistry, and examined whether MALP-2 induces COX-2 and PGE(2) by Northern blotting, RT-PCR, Western blotting and ELISA, respectively. The activation of NF-kappaB and MAP kinases (ERK1/2 and p38) was examined by Western blotting. The effects of inhibitors of NF-kappaB, MEK1/2 and p38 on MALP-2-induced PGE(2) production were also evaluated. TLR2, TLR6 and TLR4 were expressed in human placental trophoblast cells. MALP-2 significantly induced COX-2 expression and enhanced PGE(2) production in a dose-dependent manner. MALP-2 induced the activation of NF-kappaB, ERK1/2 and p38 MAPK. Inhibitors of NF-kappaB, MEK1/2 and p38 blocked MALP-2-inducible PGE(2) production. TLR2-mediated stimulation by MALP-2 induces COX-2 and PGE(2) in human placental trophoblast cells via NF-kappaB and MAP kinases pathways.

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.

Progesterone and progestational compounds attenuate tumor necrosis factor alpha-induced interleukin-8 production via nuclear factor kappa B inactivation in endometriotic stromal cells.

OBJECTIVE: To investigate whether and how P, dienogest (synthetic progestin), and danazol affected tumor necrosis factor alpha (TNFalpha)-induced interleukin-8 (IL-8) expression in endometriotic stromal cells. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. PATIENT(S): Ten patients who underwent laparoscopic surgery. INTERVENTION(S): Endometriotic stromal cells were obtained from chocolate cyst linings of the ovary. MAIN OUTCOME MEASURE(S): In the presence of TNFalpha (0.1 ng/mL) and E2 (10(-7) mol/L), the cells were cultured in medium with P (10(-6) mol/L), danazol (10(-6) mol/L), or dienogest (10(-7) mol/L). The expression of the IL-8 gene and protein was determined by Northern blotting and ELISA, respectively. Activation of nuclear factor (NF)-kappaB was evaluated by electrophoretic mobility shift assay. RESULT(S): Adding TNFalpha (0.1 ng/mL) together with E2 markedly enhanced gene and protein expression of IL-8. The up-regulation of the IL-8 gene and protein expression by TNFalpha and E2 was significantly reduced by the addition of P, dienogest, or danazol. Electrophoretic mobility shift assay revealed that incubation with TNFalpha and E2 induced NF-kappaB activation. Adding P, dienogest, or danazol attenuated NF-kappaB activation. CONCLUSION(S): The present study demonstrates for the first time that P and progestational compounds attenuate the expression of IL-8 by reducing TNFalpha-induced NF-kappaB activation in endometriotic stromal cells, suggesting a possible molecular mechanism of hormone therapy for controlling the growth 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.

Tumor necrosis factor-alpha induced the release of interleukin-6 from endometriotic stromal cells by the nuclear factor-kappaB and mitogen-activated protein kinase pathways.

OBJECTIVE: To examine the involvement of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) in the induction of interleukin-6 (IL-6) by tumor necrosis factor-alpha (TNF-alpha) in endometriotic stromal cells (ESC). DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. PATIENT(S): Twelve patients who underwent laparoscopic surgery. INTERVENTION(S): Endometriotic stromal cells were obtained from chocolate cyst linings of the ovary. MAIN OUTCOME MEASURE(S): We determined the effect of TNF-alpha on the production of IL-6 and the effect of inhibitors for NF-kappaB and the MAPK pathway on IL-6 production using ELISA. Western blottings and electrophoretic mobility shift assays were used to detect activation of NF-kappaB and extracellular signal-regulated kinase 1/2 (ERK1/2). RESULT(S): The addition of TNF-alpha (0.1 ng/mL) significantly increased IL-6 protein in endometriotic stromal cells. Western blottings and electrophoretic mobility shift assays revealed that incubation with TNF-alpha induced degradation of inhibitor kappaB (I kappaB) and expression of phosphorylated ERK1/2. The NF-kappaB inhibitor (TPCK) and MAPK inhibitor (U0126) blocked the TNF-alpha-induced IL-6 expression. Electrophoretic mobility shift assay revealed that U0126 attenuated activator protein-1 (AP-1) activation induced by TNF-alpha. CONCLUSION(S): These findings demonstrate that NF-kappaB and AP-1 activation is critical for TNF-alpha-induced IL-6 expression in endometriotic stromal cells. Novel therapeutic modalities targeting these molecules may be possible in the near future.