Akira Shirane

Resveratrol promotes expression of SIRT1 and StAR in rat ovarian granulosa cells: An implicative role of SIRT1 in the ovary

BackgroundResveratrol is a natural polyphenolic compound known for its beneficial effects on energy homeostasis, and it also has multiple properties, including anti-oxidant, anti-inflammatory, and anti-tumor activities. Recently, silent information regulator genes (Sirtuins) have been identified as targets of resveratrol. Sirtuin 1 (SIRT1), originally found as an NAD+-dependent histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. To date, the presence and physiological role of SIRT1 in the ovary are not known. Here we found that SIRT1 was localized in granulosa cells of the human ovary.MethodsThe physiological roles of resveratrol and SIRT1 in the ovary were analyzed. Immunohistochemistry was performed to localize the SIRT1 expression. SIRT1 protein expression of cultured cells and luteinized human granulosa cells was investigated by Western blot. Rat granulosa cells were obtained from diethylstilbestrol treated rats. The cells were treated with increasing doses of resveratrol, and subsequently harvested to determine mRNA levels and protein levels. Cell viability was tested by MTS assay. Cellular apoptosis was analyzed by caspase 3/7 activity test and Hoechst 33342 staining.ResultsSIRT1 protein was expressed in the human ovarian tissues and human luteinized granulosa cells. We demonstrated that resveratrol exhibited a potent concentration-dependent inhibition of rat granulosa cells viability. However, resveratrol-induced inhibition of rat granulosa cells viability is independent of apoptosis signal. Resveratrol increased mRNA levels of SIRT1, LH receptor, StAR, and P450 aromatase, while mRNA levels of FSH receptor remained unchanged. Western blot analysis was consistent with the results of quantitative real-time RT-PCR assay. In addition, progesterone secretion was induced by the treatment of resveratrol.ConclusionsThese results suggest a novel mechanism that resveratrol could enhance progesterone secretion and expression of luteinization-related genes in the ovary, and thus provide important implications to understand the mechanism of luteal phase deficiency.

Resveratrol suppresses inflammatory responses in endometrial stromal cells derived from endometriosis: A possible role of the sirtuin 1 pathway

Endometriosis is a chronic inflammatory disease. Sirtuin 1 (SIRT1) plays a role in regulation of inflammation. The role of SIRT1 in endometriosis remains unknown. We here addressed the anti‐inflammatory effects of SIRT1 on endometriosis.

SIRT3 positively regulates the expression of folliculogenesis- and luteinization-related genes and progesterone secretion by manipulating oxidative stress in human luteinized granulosa cells.

SIRT3 is a member of the sirtuin family and has recently emerged as a vital molecule in controlling the generation of reactive oxygen species (ROS) in oocytes. Appropriate levels of ROS play pivotal roles in human reproductive medicine. The aim of the present study was to investigate SIRT3 expression and analyze the SIRT3-mediated oxidative response in human luteinized granulosa cells (GCs). Human ovarian tissues were subjected to immunohistochemical analysis to localize SIRT3 expression. Hydrogen peroxide and human chorionic gonadotropin were used to analyze the relationship between ROS and SIRT3 by quantitative RT-PCR and Western blotting. Intracellular levels of ROS were investigated by fluorescence after small interfering RNA-mediated knockdown of SIRT3 in human GCs. To uncover the role of SIRT3 in folliculogenesis and luteinization, mRNA levels of related genes and the progesterone concentration were analyzed by quantitative RT-PCR and immunoassays, respectively. We detected the expression of SIRT3 in the GCs of the human ovary. The mRNA levels of SIRT3, catalase, and superoxide dismutase 1 were up-regulated by hydrogen peroxide in both COV434 cells and human GCs and down-regulated by human chorionic gonadotropin. Knockdown of SIRT3 markedly elevated ROS generation in human GCs. In addition, SIRT3 depletion resulted in decreased mRNA expression of aromatase, 17β-hydroxysteroid dehydrogenase 1, steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase in GCs and thus resulted in decreased progesterone secretion. These results have the important clinical implication that SIRT3 might play a positive role in the folliculogenesis and luteinization processes in GCs, possibly by sensing and regulating the generation of ROS. Activation of SIRT3 function might help to sustain human reproduction by maintaining GCs as well as oocytes.

Multifunctional transcription factor TFII-I is an activator of BRCA1 function

Background:The TFII-I is a multifunctional transcriptional factor known to bind specifically to several DNA sequence elements and to mediate growth factor signalling. A microdeletion at the chromosomal location 7q11.23 encoding TFII-I and the related family of transcription factors may result in the onset of Williams–Beuren syndrome, an autosomal dominant genetic disorder characterised by a unique cognitive profile, diabetes, hypertension, anxiety, and craniofacial defects. Hereditary breast and ovarian cancer susceptibility gene product BRCA1 has been shown to serve as a positive regulator of SIRT1 expression by binding to the promoter region of SIRT1, but cross talk between BRCA1 and TFII-I has not been investigated to date.Methods:A physical interaction between TFII-I and BRCA1 was explored. To determine pathophysiological function of TFII-I, its role as a transcriptional cofactor for BRCA1 was investigated.Results:We found a physical interaction between the carboxyl terminus of TFII-I and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous TFII-I and BRCA1 form a complex in nuclei of intact cells and formation of irradiation-induced nuclear foci was observed. We also showed that the expression of TFII-I stimulates the transcriptional activation function of BRCT by a transient expression assay. The expression of TFII-I also enhanced the transcriptional activation of the SIRT1 promoter mediated by full-length BRCA1.Conclusion:These results revealed the intrinsic mechanism that TFII-I may modulate the cellular functions of BRCA1, and provide important implications to understand the development of breast cancer.

Regulation of SIRT1 determines initial step of endometrial receptivity by controlling E-cadherin expression.

Sirtuin 1 (SIRT1), originally found as a class III histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. We examined the role of SIRT1 in the regulation of uterine receptivity using Ishikawa and RL95-2 endometrial carcinoma cell lines. Exogenous expression of SIRT1 significantly enhanced E-cadherin expression, while small interfering RNA-mediated depletion of endogenous SIRT1 resulted in a significant reduction of E-cadherin expression. A SIRT1 activator resveratrol elevated E-cadherin expression in a dose dependent manner, while SIRT1 repressors nicotinamide and sirtinol exhibited a dose dependent reduction of E-cadherin expression. We also showed that both forced expression of SIRT1 and activation of SIRT1 promote E-cadherin-driven reporter gene constructs, and SIRT1 is localized at E-cadherin promoter containing E-box elements in Ishikawa cells. Using an in vitro model of embryo implantation, we demonstrate that exogenous expression of SIRT1 and stimulation of SIRT1 activity resulted in the Ishikawa cell line becoming receptive to JAR cell spheroid attachment. Furthermore, resveratrol enhanced E-cadherin and Glycodelin protein expression at sites of intercellular contact, suggesting an additive role of resveratrol in promoting implantation. The initial step of human reproduction depends on the capacity of an embryo to attach and implant into the endometrial wall, and these results revealed the novel mechanism that activation and increased expression of SIRT1 play an important role in uterine receptivity.

Role of multifunctional transcription factor TFII-I and putative tumour suppressor DBC1 in cell cycle and DNA double strand damage repair

Background:In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair.Methods:Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay.Results:Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency.Conclusion:These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.

Effect of dienogest on pain and ovarian endometrioma occurrence after laparoscopic resection of uterosacral ligaments with deep infiltrating endometriosis

OBJECTIVE To evaluate the effect of dienogest (DNG) in preventing the occurrence of pain and endometriomas after laparoscopic resection of uterosacral ligaments (USLs) with deep infiltrating endometriosis (DIE). STUDY DESIGN This retrospective analysis included 126 patients who underwent laparoscopic resection of USLs with DIE followed by postoperative administration of DNG or no medication. Every 6 months postoperatively, patients answered questions and underwent ultrasound examination to identify pain and/or endometrioma. RESULT There were three (5.0%) cases of endometrioma in 59 patients from the DNG group and 21 (31.3%) cases in 67 patients from the no medication group (P=0.0002). Pain returned to preoperative levels in eight (11.9%) cases in the no medication group. No recurrence of pain occurred in the DNG group (P=0.0061). CONCLUSION The administration of DNG after resection of USLs with DIE significantly reduces the occurrence rate of endometriosis-related pain and endometriomas.

HAND2‑mediated proteolysis negatively regulates the function of estrogen receptor α

A previous study demonstrated that the progesterone‑inducible HAND2 gene product is a basic helix‑loop‑helix transcription factor and prevents mitogenic effects of estrogen receptor α (ERα) by inhibiting fibroblast growth factor signalling in mouse uteri. However, whether HAND2 directly affects the transcriptional activation function of ERα remains to be elucidated. In the present study, the physical interaction between HAND2 and ERα was investigating by performing an immunoprecipitation assay and an in vitro pull‑down assay. The results demonstrated that HAND2 and ERα interacted in a ligand‑independent manner. The in vitro pull‑down assays revealed a direct interaction between HAND2 and the amino‑terminus of ERα, termed the activation function‑1 domain. To determine the physiological significance of this interaction, the role of HAND2 as a cofactor of ERα was investigated, which revealed that HAND2 inhibited the ligand‑dependent transcriptional activation function of ERα. This result was further confirmed and the mRNA expression of vascular endothelial growth factor, an ERα‑downstream factor, was decreased by the overexpression of HAND2. This inhibition of ligand‑dependent transcriptional activation function of ERα was possibly attributed to the proteasomic degradation of ERα by HAND2. These results indicate a novel anti‑tumorigenic function of HAND2 in regulating ERα‑dependent gene expression. Considering that HAND2 is commonly hypermethylated and silenced in endometrial cancer, it is hypothesized that HAND2 may serve as a possible tumor suppressor, particularly in uterine tissue.