RESUMO
Human endometrial stromal cells (ESCs) undergo differentiation, known as decidualization, and endometrial epithelial cells mature around the embryo implantation stage. In the uterus, cyclooxygenase 2 (COX2), the rate-limiting enzyme that produces prostaglandin E2, is expressed in endometrial stromal and epithelial cells, and promotes decidualization of the former cells. Our recent study demonstrated that progesterone receptor membrane component 1 (PGRMC1) is downregulated during decidualization and may be involved in cellular senescence associated with decidualization via the transcription factor forkhead box protein O1 (FOXO1). Therefore, we investigated the role of PGRMC1 in COX2 expression during differentiation and maturation of endometrial stromal and epithelial cells. Inhibition or knockdown of PGRMC1 significantly enhanced differentiation stimuli-induced COX2 expression in both cell types. However, this COX2 expression was suppressed by FOXO1 knockdown or nuclear factor-kappa B (NF-κB) inhibition. Silencing of COX2 expression inhibited PGRMC1 knockdown-induced expression of decidual markers in ESCs. Thus, PGRMC1 may be linked to FOXO1- and NF-κB-mediated COX2 expression in endometrial cells. Taken together, our data suggest that downregulation of PGRMC1 expression facilitates differentiation of endometrial cells, i.e., decidualization and glandular maturation, via upregulation of COX2 expression.
Assuntos
Decídua , NF-kappa B , Feminino , Humanos , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Decídua/metabolismo , Endométrio , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismoRESUMO
Purpose: Extravillous trophoblasts (EVTs) invade the endometrium to establish a fetomaternal interaction during pregnancy. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) stimulate EVT invasion by binding to the EGF receptor (EGFR). We examined the role of the small GTP-binding protein Rap1 in EGF- and HB-EGF-stimulated EVT invasion. Methods: Expression of Rap1 in the first-trimester placenta was examined by immunohistochemistry. Effect of EGF or HB-EGF on Rap1 activation (GTP-Rap1) and Rap1 knockdown on invasion was assessed in EVT cell line (HTR-8/SVneo). In addition, effect of Rap1 knockdown and Rap1GAP (a Rap1 inactivator) overexpression on the activation of EGF signaling and EGFR expression were examined. Results: Rap1 was expressed by EVTs, villous cytotrophoblasts, and syncytiotrophoblasts in the placenta. EGF and HB-EGF activated Rap1 and promoted invasion of HTR-8/SVneo, and these effects were inhibited by Rap1 knockdown. The EGF- and HB-EGF-induced phosphorylation of AKT, ERK1/2, p38MAPK, and Src was inhibited by Rap1 knockdown. Furthermore, the knockdown of Rap1 reduced the EGFR protein level. Overexpression of Rap1GAP repressed EGF- and HB-EGF-induced Rap1 activation and reduced EGFR expression. Conclusion: Rap1 may function as a mediator of EGF and HB-EGF signaling pathways and can modulate EGFR expression in EVTs during placental development.
RESUMO
Uterine leiomyosarcoma is an aggressive soft tissue tumor. Stathmin, a phosphoprotein that modulates microtubule dynamics, is highly expressed in many malignancies including leiomyosarcoma. The microtubule-depolymerizing agent eribulin has been recently approved for treating malignant soft tissue tumors. Although eribulin inhibits microtubule polymerization, little is known about the relationship between eribulin treatment and stathmin dynamics. In this study, we explored the role of stathmin expression in the action of eribulin in leiomyosarcoma cells. Eribulin induced phosphorylation of stathmin and reduced expression of subunits A and C of protein phosphatase 2A (PP2A) in a leiomyosarcoma cell line. The PP2A activator FTY720 reduced levels of phosphorylated stathmin. Eribulin decreased stathmin protein levels without affecting stathmin mRNA expression. Furthermore, stathmin knockdown attenuated the inhibitory effects of eribulin on cell viability, whereas stathmin overexpression enhanced the anti-proliferative effect of eribulin. Eribulin-resistant leiomyosarcoma cell lines had enhanced expression of the class â ß-tubulin TUBB1, multi-drug resistance 1 protein MDR1 and breast cancer-resistance protein BCRP, and decreased expression of stathmin. Taken together, these results suggest that stathmin expression modulates the pharmacological efficacy of eribulin in uterine leiomyosarcoma cells.
Assuntos
Leiomiossarcoma , Estatmina , Humanos , Estatmina/genética , Estatmina/metabolismo , Estatmina/farmacologia , Leiomiossarcoma/tratamento farmacológico , Leiomiossarcoma/genética , Leiomiossarcoma/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Microtúbulos/metabolismo , Microtúbulos/patologiaRESUMO
The pathogenesis of hypertensive disorder of pregnancy (HDP), which affects about 10% of pregnant women, is still incompletely understood. Our previous study showed that endoplasmic reticulum (ER) stress influences high-temperature requirement A serine peptidase 1 (HTRA1) expression and trophoblast invasion. However, the involvement of ER stress in the regulation of HTRA subtype expression and pathophysiology of HDP has not been characterized in extravillous trophoblasts (EVTs). To investigate this, HTR8/SVneo EVTs cell line was treated with the ER stress inducers Thapsigargin (Thap) or Tunicamycin (Tuni). Treatment with either Thap or Tuni inhibited trophoblast invasion, reduced HTRA1 and HTRA3 expression, but did not alter HTRA2 or HTRA4 expression. Knockdown of HTRA1 or HTRA3 also inhibited trophoblast invasion. Furthermore, treatment with either ER stress inducer or HTRA1 silencing increased the ratio of soluble fms-like tyrosine kinase-1/placental growth factor (sFLT1/PlGF), which is a marker of HDP. Immunohistochemical analysis revealed that HTRA1 is localized to EVTs and the endometrial decidua in the placenta of patients with HDP. These results suggest that factors that cause ER stress could result in the inhibition of EVTs invasion via HTRA1.
Assuntos
Trofoblastos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Humanos , Feminino , Gravidez , Trofoblastos/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Estresse do Retículo Endoplasmático/genética , Temperatura , Fator de Crescimento Placentário , Placenta/química , Placenta/metabolismo , Movimento Celular/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/análise , Serina Endopeptidases/metabolismo , Serina Proteases/genética , Serina Proteases/metabolismoRESUMO
Eribulin, an inhibitor of microtubule dynamics, is used for treating breast cancers and sarcomas. The microtubule-destabilizing protein stathmin may modulate the antiproliferative activity of eribulin on breast cancer cells and leiomyosarcoma cells. The antitumor activity of eribulin in ovarian cancers has not been fully explored, so the present study aimed to determine the antitumor efficacy of eribulin and the involvement of stathmin in ovarian cancers. In a xenograft model of ovarian cancer, eribulin treatment reduced the tumor weight, which was accompanied by an increased level of phosphorylated stathmin. Eribulin stimulated the phosphorylation of stathmin in cultured cancer cell lines. The eribulin-induced phosphorylation of stathmin was inhibited by treatment with FTY720, an activator of protein phosphatase 2A (PP2A), and eribulin downregulated the expression of PP2A subunits. Furthermore, stathmin knockdown abrogated the inhibitory effects of eribulin on cell viability. Eribulin enhanced the antiproliferative effects of paclitaxel and concomitantly decreased stathmin expression. These results suggest that eribulin-induced phosphorylation of stathmin, mediated in part by PP2A downregulation, reduces stathmin activity and enhances the antiproliferative effects of paclitaxel in ovarian cancer. Collectively, the results of this study indicate that eribulin may suppress the proliferation of ovarian cancer cells partly by regulating the activity of stathmin.
Assuntos
Neoplasias Ovarianas , Paclitaxel , Humanos , Feminino , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Estatmina/metabolismo , Estatmina/farmacologia , Linhagem Celular Tumoral , Neoplasias Ovarianas/metabolismo , MicrotúbulosRESUMO
Decidualization - the differentiation of endometrial stromal cells (ESCs) into decidual cells - is a crucial step for successful embryo implantation and placentation that is initiated in the secretory phase of the menstrual cycle. During decidualization, ESCs undergo proliferation arrest and secrete inflammatory mediators, including senescence-associated secretory phenotype (SASP). Although several senolytic agents improve age-related diseases, their effects on cellular senescence in decidualizing ESCs has not been explored. To do this, we treated decidualized ESCs with the senolytic agents Quercetin (Que), Dasatinib (Das), and BPTES. Que decreased the number of senescence-associated ß-galactosidase (SA-ß-Gal) positive cells and expression of senescence markers in ESCs treated with the decidual stimulus (dibutyryl-cAMP plus progesterone: DP). Concomitantly, Que markedly increased the expression of the decidualization markers IGFBP1, PRL, and FOXO1, in decidualizing ESCs. Similar to Que, Das also stimulated decidualization. Treatment with a combination of Que and Das synergistically increased the expression of decidualization markers and senescence markers compared with treatment with Que or Das alone. However, BPTES did not enhance the expression of decidualization markers. These results imply that treatment with Que and/or Das can remove senescent decidual cells and enhance the decidualization of the rest of ESCs. Thus, senolytic modulation of abnormal ESC decidualization could alleviate infertility caused by dysfunctions of endometrial receptivity and embryo implantation.
Assuntos
Dasatinibe/farmacologia , Endométrio/efeitos dos fármacos , Quercetina/farmacologia , Células Estromais/efeitos dos fármacos , Sulfetos/farmacologia , Tiadiazóis/farmacologia , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Feminino , HumanosRESUMO
Previous in vitro studies have suggested that calreticulin (CALR), which is responsible for the folding and quality control of glycoproteins, may be associated with decidualization. However, its precise role in regulating decidualization has not been explored in vivo. Here, we used pregnant rat models to examine endometrial CALR expression during the peri-implantation period. We also examined whether polypectomy, a procedure that could ameliorate infertility, alters the endometrial expression levels of CALR and several implantation factors in women diagnosed as infertile. In rats, uterine CALR was expressed at a high level at the implantation site, and a marked increase in CALR expression was observed in decidual cells of normal pregnancy. In addition, endometrial CALR expression was enhanced by either administration of estradiol-17ß in the delayed implantation rat model or the artificial induction of decidualization in the pseudopregnant rat. In cultured stromal cells, siRNA-mediated silencing of CALR inhibited the decidual stimulus-induced expression of prolactin, decidual/trophoblast prolactin-related protein, and connexin 43. In humans, the endometrial expression levels of the mRNAs encoding CALR and the implantation-related factor insulin-like growth factor binding protein (IGFBP)-7 tended to increase after polypectomy. The strongest positive correlation between expression levels before polypectomy was observed for IGFBP-7 and CALR, and the strength of this correlation increased after the surgery. Thus, endometrial CALR may play a role in the formation of decidua, and the polypectomy of infertile patients may result in the co-operative expression of endometrial factors, including CALR, that could enhance endometrial receptivity.
Assuntos
Blastocisto/metabolismo , Calreticulina/genética , Decídua/metabolismo , Implantação do Embrião , Endométrio/metabolismo , Animais , Blastocisto/citologia , Calreticulina/metabolismo , Endométrio/citologia , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Infertilidade Feminina/genética , Masculino , Gravidez , Interferência de RNA , Ratos , Células Estromais/citologia , Células Estromais/metabolismo , Fatores de Tempo , Útero/metabolismoRESUMO
Protein kinase A (PKA) signalling accompanies elevated intracellular cAMP levels during endometrial stromal cell (ESC) decidualisation. Exchange protein directly activated by cAMP (EPAC), an alternate mediator of cAMP signalling, promotes PKA analogue-induced decidualisation; however, the precise mechanism by which EPAC and PKA co-operatively stimulate decidualisation has not been characterised. To examine the role of CCAAT/enhancer-binding protein (C/EBP) in EPAC- and PKA-mediated decidualisation of primary human ESCs, a reporter plasmid containing the 332bp region upstream from the transcription initiation site of the decidual prolactin (dPRL) gene was generated and the promoter activity was evaluated using a luciferase assay. The dPRL promoter activity was increased by treatment of transfected ESCs with the PKA-selective cAMP analogue N6-phenyl-cAMP (Phe) and enhanced further by co-treatment with the EPAC-selective cAMP analogue 8-(4-chlorophenyltio)-2'-O-methyl cAMP (CPT). Treatment with forskolin, an adenylyl cyclase activator, had a similar effect on reporter activity. Site-directed mutagenesis of the C/EBPß- and/or C/EBPδ-binding site in the dPRL promoter abolished Phe/CPT-mediated elevation of the reporter activity. EPAC2 knockdown markedly reduced Phe-stimulated C/EBPß and C/EBPδ mRNA levels, as well as forkhead box O1 (FOXO1) protein levels. These results suggest that EPAC signalling enhances PKA-mediated dPRL expression in ESCs by acting on C/EBP response elements in the promoter region of the gene.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Decídua/metabolismo , Endométrio/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Prolactina/metabolismo , Células Estromais/metabolismo , Ativação Transcricional/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Decídua/efeitos dos fármacos , Endométrio/citologia , Endométrio/efeitos dos fármacos , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Prolactina/genética , Regiões Promotoras Genéticas , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Infection-associated pregnancy complications cause premature delivery. Caspase-1 is involved in the maturation of interleukin (IL)-1ß, which is activated by the NLRP3 inflammasome. To characterize the significance of the NLRP3 inflammasome pathway in the placenta, the effects of activators and inhibitors on NLRP3-related molecules were examined using isolated primary trophoblasts. Caspase-1 and IL-1ß mRNA expression was markedly increased in response to lipopolysaccharide (LPS), a toll-like receptor (TLR)4 ligand. Treatment with the potassium ionophore nigericin significantly increased the level of activated caspase-1. Treatment with either LPS or nigericin stimulated IL-1ß secretion, whereas pretreatment with the ATP-sensitive K+ channel inhibitor glibenclamide, the Rho-associated coiled-coil kinase inhibitor Y-27632, or a caspase-1 inhibitor significantly decreased nigericin-induced IL-1ß secretion. In addition, dibutyryl-cAMP, which induces trophoblast differentiation, decreased expression of NLRP3, caspase-1, and IL-1ß. These findings suggest that trophoblasts can secrete IL-1ß through the NLRP3/caspase-1 pathway, which is suppressed by glibenclamide, and that the TLR4-mediated NLRP3 inflammasome pathway is more likely to be stimulated in undifferentiated than differentiated trophoblasts. Our data support the hypothesis that inhibition of the NLRP3 inflammasome can suppress placental inflammation-associated disorders.
Assuntos
Glibureto/farmacologia , Inflamassomos/fisiologia , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Trofoblastos/metabolismo , Amidas/farmacologia , Bucladesina/farmacologia , Caspase 1/metabolismo , Caspase 1/fisiologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ionóforos de Potássio/farmacologia , Gravidez , Piridinas/farmacologia , Trofoblastos/citologia , Células U937RESUMO
Microsomal prostaglandin E synthase-1 (mPGES-1) is primarily expressed in granulosa cells (GCs) in the preovulatory follicle. Both prostaglandin E2 (PGE2) and progesterone (P4) are implicated in various reproductive functions. Here, we demonstrate that mPges-1 may be a direct downstream target gene of the P4 receptor and P4-stimulated PGE2 secretion can stimulate P4 production in a newly generated mouse GC line (GtsT). Treatment of GtsT cells with a P4 receptor agonist, norgestrel, markedly increased mPGES-1 expression detected by RT-PCR analysis. PGE2 secretion measured by an enzyme-linked immunosorbent assay was enhanced by P4 treatment. Luciferase assays revealed that the proximal promoter region of the mPges-1 gene was responsible for the effects of P4 treatment. Conversely, PGE2 treatment stimulated P4 secretion, which coordinated with mRNA expression of steroidogenic acute regulatory protein. Taken together, P4 may regulate mPGES-1 expression to increase PGE2 secretion and in turn P4 production. An autocrine loop between P4 and PGE2 might function to maintain the increased levels of both in GCs.
Assuntos
Dinoprostona/metabolismo , Retroalimentação Fisiológica , Células da Granulosa/metabolismo , Progesterona/metabolismo , Prostaglandina-E Sintases/genética , Receptores de Progesterona/genética , Animais , Comunicação Autócrina , Dinoprostona/farmacologia , Feminino , Regulação da Expressão Gênica , Genes Reporter , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Microssomos/metabolismo , Norgestrel/farmacologia , Cultura Primária de Células , Progesterona/farmacologia , Regiões Promotoras Genéticas , Prostaglandina-E Sintases/metabolismo , Receptores de Progesterona/agonistas , Receptores de Progesterona/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The underlying causes of endometrial cancer (EMC) are poorly understood, and treatment options for patients with advanced stages of the disease are limited. Mutations in the phosphatase and tensin homologue gene are frequently detected in EMC. Cyclooxygenase 2 (Cox2) and mammalian target of rapamycin complex 1 (mTORC1) are known downstream targets of the phosphatase and tensin homologue protein, and their activities are up-regulated in EMC. However, it is not clear whether Cox2 and mTORC1 are crucial players in cancer progression or whether they work in parallel or cooperatively. In this study, we used a Cox2 inhibitor, celecoxib, and an mTORC1 inhibitor, rapamycin, in mouse models of EMC and in human EMC cell lines to explore the interactive roles of Cox2 and mTORC1 signaling. We found that a combined treatment with celecoxib and rapamycin markedly reduces EMC progression. We also observed that rapamycin reduces Cox2 expression, whereas celecoxib reduces mTORC1 activity. These results suggest that Cox2 and mTORC1 signaling is cross-regulated and cooperatively exacerbate EMC.
Assuntos
Carcinoma/metabolismo , Ciclo-Oxigenase 2/metabolismo , Neoplasias do Endométrio/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma/patologia , Celecoxib , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Modelos Animais de Doenças , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Pirazóis/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirolimo/farmacologia , Sulfonamidas/farmacologiaRESUMO
The optimal decidualization of endometrial stromal cells (ESCs) following embryo implantation is one of the critical steps to establish pregnancy in rodents and humans. This step is intricately regulated by ovarian hormones. Using in vitro human ESCs model, we previously showed that activation of a cAMP mediator, exchange protein directly activated by cAMP (EPAC), promotes ovarian steroid- or cAMP analog-induced decidualization. However, expressions and functions of EPAC and RAP1 in the uterus during pregnancy have not yet been examined. In this study, we found that the expression of EPAC2 and RAP1 was markedly upregulated in the decidual cells at the implantation sites on days 7 and 9 of pregnancy in rats. Furthermore, both delayed-implantation and artificial decidualization models showed that EPAC2 and RAP1 expression was enhanced in decidual cells. Significant activation of cAMP-responsive element-binding protein (CREB), a central transcriptional factor of cAMP signaling, was observed in decidual cells. These spatiotemporal expressions of protein related EPAC pathway are overlapped by sites with activated cAMP signaling, indicating the association of EPAC signaling with decidualization. Strikingly, further studies in in vitro rat decidualization model showed that the cAMP analog and medroxyprogesterone stimulated the expression of decidual markers, while knockdown of EPAC1/2 and RAP1 attenuated the expressions of these markers. Together, these findings suggest that EPAC and RAP1 are the crucial factors for endometrial decidualization in rat pregnancy.
Assuntos
Decídua/metabolismo , Implantação do Embrião , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Estromais/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Deciduoma/metabolismo , Implantação Tardia do Embrião , Feminino , Idade Gestacional , Fatores de Troca do Nucleotídeo Guanina/genética , Gravidez , Ratos , Ratos Wistar , Transdução de Sinais , Fatores de Tempo , Regulação para Cima , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Angiogenesis in the developing corpus luteum (CL) is a prerequisite for establishment and maintenance of an early pregnancy. To explore the physiological significance of insulin-like growth factor-binding protein-7 (IGFBP7) in the developing CL, the effects of IGFBP7 on vascular endothelial growth factor (VEGFA)- and luteinizing hormone (LH)-induced in vitro tube formation were tested using isolated luteal microvascular endothelial cells (LECs). Capillary-like tube formation of LECs and their proliferation were stimulated by both VEGFA and LH. IGFBP7 treatment suppressed VEGFA- or LH-induced tube formation. The proliferation and migration of LECs, and phosphorylation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase 1/2 were inhibited by IGFBP7. Furthermore, IGFBP7 attenuated VEGFA-enhanced cyclooxygenase (COX)-2 mRNA expression and prostaglandin E2 secretion. These findings suggest the possibility that luteal IGFBP7 secretion may suppress the stimulatory effect of VEGFA on angiogenesis in the early CL.
Assuntos
Corpo Lúteo/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Western Blotting , Movimento Celular , Proliferação de Células , Fatores de Crescimento Endotelial/farmacologia , Feminino , Técnicas In Vitro , Hormônio Luteinizante/farmacologia , Gravidez , Ratos , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Although preterm delivery is a major global health issue, its causes and underlying mechanism remain elusive. Using mutant mice, mimicking aspects of human preterm birth, we show here that uterine decidual senescence early in pregnancy via heightened mammalian target of rapamycin complex 1 (mTORC1) signaling is a significant contributor of preterm birth and fetal death, and that these adverse phenotypes are rescued by a low dose of rapamycin, an inhibitor of mTORC1 signaling. This role of mTORC1 signaling in determining the timing of birth in mice may help us better understand the mechanism of the timing of birth in humans and develop new and improved strategies to combat the global problem of preterm birth.
Assuntos
Nascimento Prematuro , Proteínas/metabolismo , Transdução de Sinais , Útero/metabolismo , Animais , Western Blotting , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Morte Fetal , Imuno-Histoquímica , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos , Serina-Treonina Quinases TOR , Proteína Supressora de Tumor p53/genéticaRESUMO
The fusion of mononuclear trophoblasts into multinucleate syncytiotrophoblasts is the critical event in the process of syncytialization, and its dysregulation can lead to pregnancy complications, notably hypertensive disorders of pregnancy (HDP). Oxidative stress may disrupt trophoblast syncytialization in HDP. Specifically, placentas with HDP exhibit impaired mitochondria, giving rise to the generation of reactive oxygen species (ROS) and subsequent oxidative stress. Quercetin, a bioflavonoid known for its antioxidant and anti-aging properties, has the potential to mitigate oxidative stress during trophoblast syncytialization. However, the precise mechanism underlying the action of quercetin in these processes remains to be elucidated. To explore the impact of quercetin on syncytialization, mitochondrial function, and ROS generation, cyclic AMP-stimulated BeWo cells were treated with quercetin. The expression of markers associated with cell fusion, mitochondrial function, and oxidative stress was determined using qPCR and western blotting. Additionally, morphological syncytialization and mitophagy (mitochondrial degradation) were assessed by immunofluorescence analysis. Our results revealed that quercetin increased the expression of syncytialization markers and promoted cell fusion. Furthermore, this compound also upregulated markers associated with mitophagy and mitochondrial fusion, which are corroborated by visual evidence of mitophagy through the fluorescence microscope. Cell fusion naturally stimulated ROS generation, which was attenuated by quercetin. Quercetin downregulated the expression of NRF2 and HO-1 during syncytialization, while increasing the expression of sirtuin1/3/6, which are known to play essential roles in antioxidant responses. In conclusion, quercetin effectively regulates mitochondrial function through its antioxidant properties and the suppression of ROS generation, ultimately promoting trophoblast fusion, suggesting that the flavonoid has the potential to ameliorate pregnancy-related disorder stemming from placental dysplasia.
Assuntos
Placenta , Quercetina , Gravidez , Humanos , Feminino , Placenta/metabolismo , Quercetina/farmacologia , Quercetina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Trofoblastos/metabolismo , Mitocôndrias/metabolismoRESUMO
Ovarian clear cell carcinoma (OCCC) is a unique clinicopathological subtype of epithelial ovarian cancer that is resistant to standard chemotherapy. Eribulin, a microtubule dynamics inhibitor of halichondrin class, has unique effects in the cancer microenvironment such as induction of epithelization and reduction in metastatic potential in breast cancer cells; however, nothing is known about the effect of eribulin and the detailed mechanisms in OCCC. This study aimed to investigate the involvement of ferroptosis and its mechanism in the antitumor activity of eribulin in OCCC cells and a mouse xenograft model. We found that eribulin-induced cell death was reduced by ferroptosis inhibitors; deferoxamine, an iron chelator and ferrostatin-1, a lipid peroxidation inhibitor. Eribulin increased the levels of intracellular iron, reactive oxygen species (ROS), and lipid peroxides, and increased the mitochondrial membrane potential. Eribulin downregulated the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), and superoxide dismutase (SOD) activity. The combination of eribulin and ML210, a glutathione peroxidase 4-inhibiting ferroptosis inducer, had a synergistic effect on ferroptosis. Taken together, our findings show firstly that eribulin triggers ferroptosis in OCCC and this effect occurs via the suppression of the Nrf2-HO-1 signaling pathway, SOD activity and the promotion of lipid peroxidation. These findings suggest that eribulin-induced ferroptosis is associated with its anti-tumor effect and also could be a potential therapeutic target in OCCC.
Assuntos
Carcinoma , Ferroptose , Furanos , Cetonas , Policetídeos de Poliéter , Humanos , Camundongos , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/farmacologia , Microambiente TumoralRESUMO
Mononuclear cytotrophoblasts (CTs) differentiate and fuse to form multinuclear syncytiotrophoblasts (STs), which produce human chorionic gonadotropin (hCG) and progesterone to maintain pregnancy. Impaired differentiation and fusion of CTs to form STs are associated with hypertensive disorders of pregnancy and fetal growth restriction. Progesterone receptor membrane component 1 (PGRMC1) is a multifunctional single transmembrane heme-binding protein. We previously demonstrated that downregulation of PGRMC1 promotes endometrial stromal cell differentiation (decidualization). Here, we explored the role of PGRMC1 in trophoblast differentiation and fusion. PGRMC1 expression was lower in STs than in CTs of first-trimester placental tissues. PGRMC1 expression in BeWo cells (a trophoblast-derived choriocarcinoma cell line) decreased upon dibutyryl-cAMP (db-cAMP)-induced differentiation. Both inhibition and knockdown of PGRMC1 stimulated hCG production in the presence of db-cAMP. Furthermore, a quantitative cell fusion assay we developed revealed that inhibition and knockdown of PGRMC1 enhanced db-cAMP-stimulated cell fusion. Peroxisome proliferator-activated receptor γ (PPARγ) agonists decreased PGRMC1 expression and stimulated the cell fusion in BeWo cells. These findings suggest that downregulation of PGRMC1 expression in part through activation of PPARγ during trophoblast differentiation promotes hCG production and cell fusion for formation and maintenance of placental villi during pregnancy.
Assuntos
PPAR gama , Placenta , Humanos , Feminino , Gravidez , Regulação para Baixo , PPAR gama/metabolismo , Placenta/metabolismo , Linhagem Celular , Gonadotropina Coriônica/farmacologia , Trofoblastos/fisiologia , Diferenciação Celular/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismoRESUMO
Heightened mammalian target of rapamycin complex 1 (mTORC1) activity by genetic deletion of its direct inhibitor, Tsc1, is associated with aberrant development and dysfunction of the female reproductive tract in mice. Here, we compared the phenotypes of mice with conditional deletion of Tsc1 in the female reproductive tract by either progesterone receptor (PR)-Cre (Tsc1(PR(d/d))), which inactivates Tsc1 in all major cell types in the uterus (epithelium, stroma and myometrium), or anti-Mullerian hormone type 2 receptor (Amhr2)-Cre (Tsc1(Amhr2(d/d))), which inactivates stromal and myometrial Tsc1. Tsc1(PR(d/d)) and Tsc1(Amhr2(d/d)) females are infertile resulting from oviductal hyperplasia, retention of embryos in the oviduct and implantation failure. In contrast to the appropriate embryonic development after fertilization seen in Tsc1(Amhr2(d/d)) females, embryo development was disrupted in Tsc1(PR(d/d)) females. In addition, uteri in Tsc1(PR(d/d)) and Tsc1(Amhr2(d/d)) females showed epithelial hyperplasia but not endometrial cancer. In conclusion, Tsc1(PR(d/d)) and Tsc1(Amhr2(d/d)) have overlapping yet distinct phenotypes in the context of compartment-specific deletion of Tsc1.
Assuntos
Complexos Multiproteicos/metabolismo , Oviductos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Útero/metabolismo , Animais , Estradiol/sangue , Feminino , Fertilização in vitro , Imuno-Histoquímica , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Mutantes , Complexos Multiproteicos/genética , Ovário/metabolismo , Progesterona/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/genética , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
The appropriate differentiation of endometrial stromal cells (ESCs) into decidual cells is required for embryo implantation and subsequent placentation into humans. Decidualization is accompanied by the appearance of senescent-like cells. We recently reported the secretory phase-specific downregulation of endometrial progesterone receptor membrane component 1 (PGRMC1) and enhanced decidualization upon PGRMC1 knockdown and inhibition in cultured ESCs. However, it remains unknown whether PGRMC1 is involved in cellular senescence during decidualization. Here, we showed that the small interfering RNA (siRNA)-mediated knockdown of PGRMC1 and the inhibition of PGRMC1 by AG-205 increased the expression of the transcription factor forkhead box protein O1 (FOXO1) and the senescence-associated ß-galactosidase activity in cAMP analog- and progesterone-treated ESCs. Furthermore, the knockdown of FOXO1 repressed the decidual senescence induced by siRNA-based PGRMC1 knockdown or AG-205 treatment. Taken together, the decreased PGRMC1 expression in ESCs may accelerate decidualization and cellular senescence via the upregulation of FOXO1 expression for appropriate endometrial remodeling and embryo implantation during the secretory phase.
Assuntos
Decídua , Proteína Forkhead Box O1/genética , Receptores de Progesterona/metabolismo , Células Estromais , Células Cultivadas , Senescência Celular , Decídua/metabolismo , Feminino , Proteína Forkhead Box O1/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Gravidez , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Células Estromais/metabolismoRESUMO
Endometriosis is characterized by the presence of inflamed and fibrotic endometrial tissue outside the uterine cavity. Previously, we found decreased SERPINA1 (alpha-1 antitrypsin) expression in endometriosis-like lesions in a mouse model of endometriosis, suggesting that it exacerbated inflammation in these lesions. However, the molecular mechanism(s) by which SERPINA1 affects expression of inflammatory factors and development of endometriotic lesions have not been fully characterized. To investigate the role of intracellular SERPINA1 in endometrial stromal cells (ESCs), we performed RNA sequence analysis using RNA extracted from ESCs in which SERPINA1 was knocked down. The analysis identified several toll-like receptor (TLR)-related factors as being upregulated. Silencing of SERPINA1 increased expression of TLR3 and TLR4 in ESCs, as well as several TLR signaling pathway components, including MYD88, IRAK1/4, interleukin (IL)-1ß, and interferon (IFN)-ß. TLR3 or TLR4 agonists increased expression of inflammatory factors in SERPINA1-knockdown ESCs, whereas TLR3 or TLR4 inhibitors decreased expression. In addition, treatment with recombinant IL-1ß or IFN-ß increased expression of MYD88 and inflammatory factors in ESCs. Immunohistochemical analysis of endometriotic tissues showed that TLR3, TLR4, and MYD88 were localized in endometriosis lesions. Taken together, the data suggest that reduced expression of SERPINA1 induces expression of inflammatory factors by ESCs, which in turn are associated with TLR3/4, IL-1ß, and IFN-ß signaling. Regulation of intracellular SERPINA1 levels in ESCs may be a strategy to inhibit inflammatory responses in endometriotic lesions.