Relaxin-2 May Suppress Endometriosis by Reducing Fibrosis, Scar Formation, and Inflammation.

BACKGROUND: Relaxin (RLX)-2, produced by the corpus luteum and placenta, is known to be potentially effective in fibrotic diseases of the heart, lungs, kidneys, and bladder; however, its effectiveness in endometriosis has not yet been investigated. In the present study, we conducted a comprehensive study on the effect of RLX-2 on endometriosis. We checked the expressions of LGR-7, a primary receptor of RLX-2, in endometriomas using immunohistochemistry. Endometriotic stromal cells (ESCs) purified from surgical specimens were used in in vitro experiments. The effects of RLX-2 on ESCs were evaluated by quantitative-PCR, ELISA, and Western blotting. Gel contraction assay was used to assess the contraction suppressive effect of RLX-2. The effect of RLX-2 was also examined in the endometriosis mouse model. LGR-7 was expressed in endometriotic lesions. In ESCs, RLX-2 increased the production of cAMP and suppressed the secretion of interleukin-8, an inflammatory cytokine, by 15% and mRNA expression of fibrosis-related molecules, plasminogen activator inhibitor-1 (PAI-1), and collagen-I by approximately 50% (p < 0.05). In the gel contraction assay, RLX-2 significantly suppressed the contraction of ESCs, which was cancelled by removing RLX-2 from the medium or by adding H89, a Protein Kinase A (PKA) inhibitor. In ESCs stimulated with RLX-2, p38 MAPK phosphorylation was significantly suppressed. In the endometriosis mouse model, administration of RLX-2 significantly decreased the area of the endometriotic-like lesion with decreasing fibrotic component compared to non-treated control (p = 0.01). RLX-2 may contribute to the control of endometriotic lesion by suppressing fibrosis, scar formation, and inflammation.

Sphingosine 1 Phosphate (S1P) Increased IL-6 Expression and Cell Growth in Endometriotic Cells.

OBJECTS: There is growing evidence that sphingosine 1-phosphate (S1P) is involved in inflammatory diseases. As endometriosis is known as an inflammatory disease, we investigated the role of S1P system in the development of endometriosis. METHODS: The expression of sphingosine kinase (SphK) 1 in endometriosis lesions was examined by immunohistochemistry. The cystic fluid of ovarian cysts/tumors were obtained to measure S1P concentrations. Endometriotic stromal cells (ESC) derived from endometrioma were used for in vitro experiments. RESULTS: Sphingosine kinase 1 was detected in epithelium and stromal cells of endometriotic lesions. The mean S1P concentration in the cystic fluid of endometriomas was higher than that in nonendometriomas significantly (98.2 nM vs less than 1.5 nM, P < .01). Interleukin-1ß (IL-1ß) or transforming growth factor-ß exhibited 2.7-fold and 11.5-fold increase in SphK1 messenger RNA (mRNA) expression in ESC, respectively (P < .01). Higher dose of S1P (125nM) increased the cell number of ESC by 20%, and low dose of S1P (1.25 nM and 12.5 nM) induced IL-6 mRNA production and IL-6 secretion by ESC dose-dependently. JTE013, an antagonist for S1PR2, partially suppressed IL-6 induction by S1P (P < .05). JTE013 and VPC23019, an antagonist for S1PR1 and S1PR3, suppressed the ESC proliferation induced by S1P. CONCLUSION: The present study for the first time proved that the SphK-S1P-S1PR axis play a role of accelerating inflammation and growth of endometriotic cells.

RSK-MASTL Pathway Delays Meiotic Exit in Mouse Zygotes to Ensure Paternal Chromosome Stability.

During vertebrate fertilization, sperm chromatin remodeling occurs concomitantly with maternal chromosome segregation at anaphase II, leading to simultaneous formation of two pronuclei. In mammals, these processes take much longer than in other vertebrates. Here, we explore the molecular basis and physiological importance of this mammalian-specific temporal regulation using mouse oocytes. We demonstrate the involvement of protein phosphatase in temporal regulation. Early onset of pronuclear formation causes paternal-biased abnormalities in pronuclear morphology and chromosome segregation at the first mitosis. After oocyte activation, CDK1-MASTL-ENSA, a protein phosphatase 2A-suppressive pathway, remains active despite the absence of cyclin B and contributes to delayed pronuclear formation. Sustained activation of MASTL involves ribosomal S6 kinase (RSK)-mediated phosphorylation of Thr297, which is conserved only among mammalian MASTLs. Our findings reveal the role of RSK in mouse oocytes, showing that the RSK-MASTL pathway allows mammalian-specific prolonged meiotic exit and ensures the faithful conversion from sperm to paternal pronuclei.

PAI-1 in granulosa cells is suppressed directly by statin and indirectly by suppressing TGF-ß and TNF-α in mononuclear cells by insulin-sensitizing drugs.

PROBLEM: Plasminogen activator inhibitor-1 (PAI-1) is elevated in women with polycystic ovary syndrome (PCOS), but the regulation in granulosa cells (GCs) is unclear. METHOD OF STUDY: PAI-1 expression in PCOS ovaries was investigated immunohistologically. PAI-1 expressions in HGrC1, a human GC cell line, were investigated at mRNA and activity levels. The expressions of TGF-ß and TNF-α in peritoneal fluid mononuclear cells (PFMCs) were measured with quantitative PCR. RESULTS: Little PAI-1 expression is observed in healthy GCs, whereas GCs of PCOS and atretic follicle exhibit distinct expression in vivo. In vitro study using HGrC1 shows that TGF-ß and TNF-α increase PAI-1 mRNA and its activity, and both together exhibit a synergistic effect. The expression of PAI-1 mRNA is suppressed by simvastatin. Moreover, insulin-sensitizing drugs (metformin, pioglitazone, and rosiglitazone) suppress LPS-induced TGF-ß and TNF-α mRNA expression in PFMC. CONCLUSION: Statin and insulin-sensitizing drugs may provide a potential therapy for PCOS via down-regulation of PAI-1 expression in GCs and down-regulation of TGF-ß and TNF-α expression in PFMC, respectively.

Oral contraceptive therapy reduces serum relaxin-2 in elite female athletes.

AIM: Recent investigations have demonstrated that athletes with high relaxin-2 levels have a high risk of anterior cruciate ligament injuries, while athletes taking oral contraceptives (OC) have low relaxin-2 levels. It has not yet been clarified whether taking OC reduces relaxin-2 levels. The purpose of this study was to investigate changes in relaxin-2 levels in athletes taking OC. METHODS: Levels of relaxin-2, estradiol, progesterone, luteinizing hormone and follicle-stimulating hormone were measured in serum samples (n = 183) from 106 elite female athletes. Five athletes with serum relaxin-2 concentrations > 6 pg/mL during the luteal phase were recruited to assess the effect of OC therapy. RESULTS: Serum relaxin-2 concentrations were significantly higher during the luteal phase (n = 57) than in the follicular phase (n = 72), or in athletes on OC therapy (n = 10) (P < 0.001, P < 0.001 and P < 0.05, respectively). In the luteal phase, 36.8% (21/57) of the athletes had relaxin levels > 6 pg/mL. In 23 athletes, serum relaxin-2 concentrations were measured during both the follicular and luteal phases, revealing that relaxin-2 levels were significantly higher in the luteal phase compared with the follicular phase. In 5 out of 23 athletes, serum relaxin-2 concentrations were > 6 pg/mL in the luteal phase and during the second cycle of OC therapy, relaxin-2 concentrations decreased dramatically to below the detection limit (0.26 pg/mL). CONCLUSIONS: High serum relaxin-2 concentrations were only detected during the luteal phase. In athletes with high relaxin-2 concentrations during the luteal phase, OC therapy decreased serum relaxin-2 levels.

Alpha-7 nicotinic acetylcholine receptor (nAChR) agonist inhibits the development of endometriosis by regulating inflammation.

OBJECTIVE: We investigated α-7 nAchR expression in human peritoneal macrophages and examined whether activation of nAchR might be a new therapy for endometriosis. MATERIALS AND METHODS: Human peritoneal fluid mononuclear cells (PFMC) were stimulated with lipopolysaccharide (LPS) in the presence of α-7 nAChR agonists. In a murine endometriosis model, α-7 nAChR modulators were administered. RESULTS: Human PFMC expressed α-7 nAChR at the mRNA and protein levels. Activation of α-7 nAChR with its agonists led to significant (P<.01) suppression of LPS-induced interleukin (IL) -1ß expression. In a murine endometriosis model, one week after inoculation of endometrium to the peritoneal cavity, α-7 nAChR agonist significantly suppressed the expression of IL-1ß mRNA (P<.01), which was negated when α-7 nAChR antagonist was administered simultaneously. α-7 nAChR agonist significantly suppressed the formation of endometriotic lesions, which was reversed with α-7 nAChR antagonist. CONCLUSION: Activation of nAChR might be a new candidate for treatment of endometriosis.

The microtubule-binding and coiled-coil domains of Kid are required to turn off the polar ejection force at anaphase.

Mitotic chromosomes move dynamically along the spindle microtubules using the forces generated by motor proteins such as chromokinesin Kid (also known as KIF22). Kid generates a polar ejection force and contributes to alignment of the chromosome arms during prometaphase and metaphase, whereas during anaphase, Kid contributes to chromosome compaction. How Kid is regulated and how this regulation is important for chromosome dynamics remains unclear. Here, we address these questions by expressing mutant forms of Kid in Kid-deficient cells. We demonstrate that Cdk1-mediated phosphorylation of Thr463 is required to generate the polar ejection force on Kid-binding chromosomes, whereas dephosphorylation of Thr463 prevents generation of the ejection force on such chromosomes. In addition to activation of the second microtubule-binding domain through dephosphorylation of Thr463, the coiled-coil domain is essential in suspending generation of the polar ejection force, preventing separated chromosomes from becoming recongressed during anaphase. We propose that phosphorylation of Thr463 switches the mitotic chromosome movement from an anti-poleward direction to a poleward direction by converting the Kid functional mode from polar-ejection-force-ON to -OFF during the metaphase-anaphase transition, and that both the second microtubule-binding domain and the coiled-coil domain are involved in this switching process.