RESUMO
Preeclampsia is a gestational disease characterized by two major pathological changes-shallow trophoblast invasion and impaired spiral artery remodeling. Atrial natriuretic peptide (ANP) is a kind of peptide hormone that regulates blood pressure, while the lack of active ANP participates in preeclampsia pathogenesis. However, the underlying mechanism of how ANP modulates trophoblasts function remains unclarified. Here, we performed isobaric tags for relative and absolute quantification (iTRAQ) in ANP-treated HTR-8/SVneo cells and identified Protein Kinase 3 (PKN3) as the downstream factor of ANP, which was downregulated in preeclamptic placenta. Chromatin immunoprecipitation analysis and luciferase assays showed that NFYA was one of the transcription factors for the PKN3 promoter, which was also regulated by ANP treatment in HTR-8/SVneo cells. Transmission electron microscopy and Western Blotting in HTR-8/SVneo cells indicated that ANP inhibited autophagy via AMPK-mTORC1 signaling, while excess autophagy was observed in preeclamptic placenta. The increased expression of PKN3 and enhanced cell invasion ability in HTR-8/SVneo cells induced by ANP could be abolished by autophagy activation or transfection with PKN3 shRNA or NFYA shRNA or NPR-A shRNA via regulating the invasion-related genes and the epithelial mesenchymal transition molecules. Our results demonstrated that ANP could enhance trophoblast invasion by upregulating PKN3 via NFYA promotion through autophagy inhibition in an AMPK/mTORC1 signaling-dependent manner.
Assuntos
Pré-Eclâmpsia , Feminino , Humanos , Gravidez , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Linhagem Celular , Movimento Celular , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , RNA Interferente Pequeno/metabolismo , Trofoblastos/metabolismo , Fator Natriurético AtrialRESUMO
Deficiency of decidual NK (dNK) cell number and function has been widely regarded as an important cause of spontaneous abortion. However, the metabolic mechanism underlying the crosstalk between dNK cells and embryonic trophoblasts during early pregnancy remains largely unknown. Here, we observed that enriched glutamine and activated glutaminolysis in dNK cells contribute to trophoblast invasion and embryo growth by insulin-like growth factor-1 (IGF-1) and growth differentiation factor-15 (GDF-15) secretion. Mechanistically, these processes are dependent on the downregulation of EGLN1-HIF-1α mediated by α-ketoglutarate (α-KG). Blocking glutaminolysis with the GLS inhibitor BPTES or the glutamate dehydrogenase inhibitor EGCG leads to early embryo implantation failure, spontaneous abortion and/or fetal growth restriction in pregnant mice with impaired trophoblast invasion. Additionally, α-KG supplementation significantly alleviated pregnancy loss mediated by defective glutaminolysis in vivo, suggesting that inactivated glutamine/α-ketoglutarate metabolism in dNK cells impaired trophoblast invasion and induced pregnancy loss.
Assuntos
Aborto Espontâneo , Animais , Feminino , Camundongos , Gravidez , Diferenciação Celular , Glutamina/farmacologia , Fator 15 de Diferenciação de Crescimento , Fator de Crescimento Insulin-Like I , Ácidos Cetoglutáricos/farmacologiaRESUMO
During gestation, excess palmitate (PA) is enriched in decidua. Both excess PA and decidual dysfunctions are associated with numerous adverse pregnancy outcomes such as gestational diabetes, preeclampsia and preterm birth and intrauterine growth restriction. Here, mRNA data about the effects of PA were collected from multiple databases and analyzed. Human decidual tissues were obtained from clinically normal pregnancies, terminated for non-medical reasons, during the first trimester, and decidual stromal cells (DSCs) were isolated and exposed to PA, alone or together with the inhibitors of Toll-like receptor 4 (TLR4), Jun N-terminal kinase (JNK), nuclear factor-kappa-gene binding (NF-kB) or glutamine (GLN) oxidation. Furthermore, DSCs were transfected with lentiviral particles overexpressing human TLR4. We demonstrate that excess PA interacting with its receptor TLR4 disturbs DSC hemostasis during the first trimester. Specifically, high PA signal induced DSC apoptosis and formed an inflammatory program (elevated interleukin-1 beta and decreased interleukin-10) via the activation of TLR4/JNK/NF-kB pathways. A complexed cross-talk was found between TLR4/JNK/NF-kB signals and PA deposition in DSCs. Besides, under an excess PA environment, GLN oxidation was significantly enhanced in DSCs and the suppression of GLN oxidation further augmented PA-mediated DSC apoptosis and inflammatory responses. In conclusion, excess PA induces apoptosis and inflammation in DSCs via the TLR4/JNK/NF-kB pathways, which can be augmented by the suppression of GLN oxidation.
Assuntos
Apoptose/efeitos dos fármacos , Decídua/citologia , Glutamina/metabolismo , Sistema de Sinalização das MAP Quinases , NF-kappa B/fisiologia , Células Estromais/efeitos dos fármacos , Receptor 4 Toll-Like/fisiologia , Feminino , Ontologia Genética , Humanos , Oxirredução , Palmitatos/farmacologia , Gravidez , Primeiro Trimestre da Gravidez , Proteínas Recombinantes/metabolismo , Células Estromais/citologia , Receptor 4 Toll-Like/genética , TransfecçãoRESUMO
BACKGROUND: Trophoblast cells are required for the establishment of pregnancy and fetal development. Apoptosis is an essential feature for trophoblast invasion. Uncontrolled trophoblast apoptosis is related to some complicate pregnancies. Oxidative stress (OS) is an important inducer of trophoblast apoptosis. Cyclosporin A (CsA) has been shown to promote the activity of trophoblast cells and reduce OS-induced oxidative injury. We investigated the role and mechanism of CsA in oxidative stress-induced trophoblast cell apoptosis. METHODS: JEG-3 cells were cocultured with H2O2 and CsA. Cell viability and morphology were measured by MTT assay and DAPI staining. Cell apoptosis was tested with annexin V/PI staining. The expression of Bcl-2-associated X protein (Bax), B-cell lymphoma/leukemia-2 (Bcl-2), cleaved poly (ADP-ribose) polymerase (PARP) and pro-caspase-3 was assayed by western blotting. The protein expression and phosphorylation of p53 and mitogen-activated protein kinase (MAPK) kinases (JNK, ERK1/2 and p38) were examined by western blotting. RESULTS: CsA increased the viability, alleviated morphological injury and reduced cell apoptosis of the H2O2-treated JEG-3 cells. CsA also attenuated the activation of p53, decreased the expression of Bax and cleavage of PARP, and increased the expression of Bcl-2 and pro-caspase-3 in the JEG-3 treated with H2O2. Furthermore, CsA reduced the activation of JNK and P38 but had no significant effect on the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the H2O2-treated JEG-3 cells. Promoting the activation of JNK and p38 impaired the protective effect of CsA on OS-induced trophoblast apoptosis. CONCLUSIONS: These results suggested that CsA protected trophoblast cells from OS-induced apoptosis via the inhibition of the p53 and JNK/p38 signaling pathways.
Assuntos
Apoptose/efeitos dos fármacos , Ciclosporina/farmacologia , Trofoblastos/efeitos dos fármacos , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Gravidez , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trofoblastos/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Adrenergic receptor (AR), one of the key receptors for nervous system, plays an important role in the immune microenvironment and the progression of many diseases. In recent years, the regulation of ARs and its signal on macrophages has become a research hotspot. Researchers found that ARs could exert different regulatory functions on macrophages in different microenvironments, which in turn affects occurrence and development of diseases such as tumor, heart failure, obesity, acute injury, infection and pregnancy-related diseases. This review summarizes the expression and functional regulation of ARs on macrophages, and the role of ARs in microenvironment of related diseases, which might provide new ideas for the treatments.
Assuntos
Doença , Macrófagos/fisiologia , Receptores Adrenérgicos/fisiologia , Transdução de Sinais , HumanosRESUMO
BACKGROUND/AIMS: Uterus endometrial cancer (UEC) is the common malignancy among gynecologic cancers, and most of them are type I estrogen-dependent UEC. Diabetes is well-known risk factor for the development of UEC. However, the underlying link between high glucose (HG) and the estrogen receptor in UEC remains unclear. Epithelial-mesenchymal transition (EMT) has also been shown to occur during the initiation of metastasis in cancer progression. The aim of this study was to determine the relationships and roles of HG, estrogen receptor and EMT in the growth and migration of UEC. METHODS: The expression of glucose transport protein 4 (GLUT4) in the control endometrium and UEC tissues was detected by immunohistochemistry (IHC); the cell viability and invasion were analyzed through CCK-8 and Matrigel invasion assays; the transcriptional level of EMT-related genes was evaluated through real-time PCR; and the effect of HG and / or GLUT4 on estrogen receptors, vascular endothelial growth factor (VEGF) and its receptor VEGFR was analyzed through western blotting, ELISA and flow cytometry (FCM) assay, respectively. In addition, Ishikawa-xenografted nude mice were constructed and were used to analyze the effect of estrogen and GLUT4 on the growth of UEC in vivo. RESULTS: Here, we found that exposure to HG led to a high level of viability and invasion of UEC cell lines (UECC, Ishikawa and RL95-2 cells). Compared with the normal endometrium, a higher level of GLUT4 was observed in UEC tissues. Silencing GLUT4 obviously inhibited the HG-promoted viability, invasion and expression of EMT-related genes (TWIST, SNAIL and CTNNB1) of UECC promoted by HG. Further analysis showed that HG and GLUT4 promoted the secretion of VEGF and expression of VEGFR in UECC. Treatment with HG led to the increase of estrogen receptor α (ERα) and ß (ERß) in UECC, blocking ERα or ERß resulted in the decreases in GLUT4 expression, TWIST, SNAIL and CTNNB1 transcription, and VEGF and VEGFR expression in UECC. Treatment with anti-human VEGF neutralizing antibody restricted the viability and invasion of UECC that was induced by HG and estrogen. Exposure to estrogen accelerated growth, VEGF production, and TWIST and CTNNB1 expression in UEC in Ishikawa-xenografted nude mice, and silencing GLUT4 restricted these effects. CONCLUSION: These data suggest that HG increases GLUT4 and VEGF/VEGFR expression, further promotes EMT process and accelerates the development of UEC by up-regulating ER.
Assuntos
Neoplasias do Endométrio/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transportador de Glucose Tipo 4/metabolismo , Glucose/farmacologia , Receptores de Estrogênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/metabolismo , Feminino , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/genética , Humanos , Camundongos , Camundongos Nus , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição Twist/genética , Fatores de Transcrição Twist/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Recurrent spontaneous abortion (RSA) is a common health problem that affects about 5% of fertile women, when it occurs for unknown reasons, it is called unexplained recurrent spontaneous abortion (URSA). Traditional Chinese medicine, such as Bu-Shen-Yi-Qi formula which consists of Dangshen, Tusizi, Baizhu, Baishuo, Duzhong, Sangjisheng, Sugeng, and Tiaohuangqin, has played an invaluable role in the treatment of RSA since ancient times. However, the mechanism of how it takes effect is still not clear. To identify Bu-Shen-Yi-Qi formula could modulate immune condition at maternal-fetal interface via its effect on trophoblasts, HTR-8 of different treatment were co-cultured with peripheral or decidual natural killer (NK) cells, and the receptors such as NKP30 and NKP46 expression on NK cells were measured by flow cytometry (FCM). In this study, we found that herb medium could increase the IDO expression at appropriate concentrations. As an inhibitor of IDO, 1-MT could impair the inhibitory function of trophoblasts on NK cells. Furthermore, Bu-Shen-Yi-Qi formula could enhance the inhibitory function of trophoblasts on NK cells. In conclusion, Bu-Shen-Yi-Qi formula can inhibit NK cytotoxicity by up-regulating IDO expression in trophoblasts and play a role in the treatment of URSA patients.
Assuntos
Aborto Habitual/tratamento farmacológico , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Medicina Tradicional Chinesa , Trofoblastos/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Feminino , Humanos , Gravidez , Trofoblastos/enzimologiaRESUMO
Decidual NK (dNK) cells, identified as CD56brightCD16-CD3-, account for ~70% of lymphocytes within the uterine wall during early pregnancy. Accumulating evidence suggests that tight interactions between placental trophoblasts and dNK cells are critical for trophoblast cell differentiation. However, the underlying mechanism remains to be explored in detail. In the present study, conditioned medium (CM) was collected from cultured primary human dNK cells. Primary cytotrophoblasts (CTBs) or the human trophoblast cell line HTR8/SVneo was treated with dNK-CM and co-cultured with human umbilical vein endothelial cells (HUVECs) in a three-dimensional Matrigel scaffold, and the formation of tube structures was dynamically monitored with live cell imaging. Trophoblast invasion was analyzed with a transwell invasion assay. The data demonstrated that the treatment of HTR8/SVneo cells or CTBs with dNK-CM remarkably promoted trophoblast invasion and tube formation in the presence of HUVECs. The epithelial marker E-cadherin was reduced, while the expression of endothelial markers NCAM, VE-cadherin and integrin ß1 was significantly promoted in the HTR8/SVneo cells upon treatment with dNK-CM. Antibody blocking experiments revealed that the dNK cells promoted trophoblast invasion through the production of IL-8 and HGF, and they induced trophoblast differentiation toward endothelial phenotype by producing VEGF-C and HGF. These results provide new evidence to clarify the finely tuned interactions between trophoblasts and dNK cells at the maternal-fetal interface.
Assuntos
Decídua/imunologia , Células Endoteliais/imunologia , Fator de Crescimento de Hepatócito/metabolismo , Células Matadoras Naturais/imunologia , Trofoblastos/imunologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Antígeno CD56/metabolismo , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-8/metabolismo , Morfogênese , Gravidez , Cultura Primária de CélulasRESUMO
STUDY QUESTION: Do regulatory T cells (Tregs) contribute to angiogenesis in endometriosis? SUMMARY ANSWER: High levels of CCL17 and CCL22 cause the recruitment of Tregs, upregulate the immunosuppression of Tregs and, in turn, may promote angiogenesis in endometrial cells in synergy with proinflammatory cytokines. WHAT IS ALREADY KNOWN: The peritoneal fluid of patients with endometriosis has a higher percentage of Tregs than that of normal individuals; however, the regulatory role of Tregs in the disease remains unclear. STUDY DESIGN, SIZE, DURATION: This study used primary human endometrial stromal cells (ESCs), monocytes (Mo), Tregs and human umbilical vein endothelial cells (HUVECs). All experiments were performed at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: The migration of Tregs was evaluated by the transwell migration assay. The activation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase and p38 signaling pathways was examined using the In-Cell WesternTM (LI-COR®) western blot analysis system, as well as by traditional western blot analysis. Changes in the expression of CCL22, CCL17, transforming growth factor-beta 1 (TGF-ß1), Interleukin (IL)-1ß, tumor necrosis factor alpha (TNF-α), IL-8 and vascular endothelial growth factor (VEGF) in cell-culture supernatant were detected by ELISA. We analyzed the Tregs by multicolor flow cytometry to directly test the expression of CCR4, CD4, CD25, Foxp3, CTLA-4, CD39 and CD73. MAIN RESULTS AND THE ROLE OF CHANCE: Our results showed that ESCs-Mo co-culture produced significantly higher levels of CCL22 and CCL17 than ESCs or Mo cultured alone, and that estradiol (E2) or progesterone (P) further promoted this upregulation, demonstrating stronger chemotaxis on Tregs. The co-culture of ESCs with Mo stimulated TGF-ß1 secretion by Tregs, which could be inhibited by anti-CCL22 or/and anti-CCL17 neutralizing antibodies (Abs). The expression of CCR4 by Tregs was upregulated in ESCs-Mo co-culture, especially by treatment with E2 and/or P, and this effect could be abolished by anti-CCL22 and/or anti-CCL17-neutralizing Abs. The Treg-ESCs-Mo co-culture treated with E2 (10-8 mol/l) and P (10-8 mol/l) could enhance the immunosuppression of Tregs, as proved by the elevated expression of Foxp3, CTLA-4, CD39 and CD73 on Tregs. ESCs-Mo co-culture could significantly promote the secretion of IL-1ß and TNF-α. TGF-ß1 from Tregs could activate p38/ERK1/2 signaling pathways in ESCs, and IL-1ß and TNF-α produced by ESCs-Mo co-culture had synergistic roles with TGF-ß1. TGF-ß1 and the proinflammatory cytokines IL-1ß or TNF-α could synergistically promote IL-8 and VEGF expression in ESCs via the p38/ERK1/2 signaling pathways. The high levels of IL-8 and VEGF in the supernatant of ESCs stimulated the angiogenesis of HUVECs. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This study was only performed in vitro using eutopic ESCs, instead of ectopic cells, from endometriosis patients. Therefore, it is necessary to do further experiments to determine whether Tregs promote angiogenesis in the endometriotic milieu in synergy with proinflammatory cytokines in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Co-targeting Tregs and proinflammatory cytokines may be an effective treatment for endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Ministry of Science and Technology of China 2015CB943300 to L.D.-J.; National Natural Science Foundation of China, item number 81200425 to W.X.-Q.; National Natural Science Foundation of China, item number 81471548 to L.D.-J.; and The Research Fund for the Doctoral Program of Higher Education of China to W.X.-Q. (20110071120093). The authors have no conflicts of interest to declare.
Assuntos
Quimiocina CCL17/metabolismo , Quimiocina CCL22/metabolismo , Endometriose/patologia , Endométrio/patologia , Endotélio Vascular/patologia , Neovascularização Patológica/patologia , Linfócitos T Reguladores/patologia , Adulto , Biomarcadores/metabolismo , Comunicação Celular , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Endometriose/imunologia , Endometriose/metabolismo , Endométrio/irrigação sanguínea , Endométrio/imunologia , Endométrio/metabolismo , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Terapia de Imunossupressão , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Células Estromais/imunologia , Células Estromais/metabolismo , Células Estromais/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
The dysfunction of NK cells in women with endometriosis (EMS) contributes to the immune escape of menstrual endometrial fragments refluxed into the peritoneal cavity. The reciprocal communications between endometrial stromal cells (ESCs) and lymphocytes facilitate the development of EMS. However, the mechanism of these communications on cytotoxicity of natural killer (NK) cells in endometriotic milieus is still largely unknown. To imitate the local immune microenvironment, the co-culture systems of ESCs from patients with EMS and monocyte-derived macrophages or of ESCs, macrophages and NK cells were constructed. The cytokine levels in the co-culture unit were evaluated by ELISA. The expression of functional molecules in NK cells was detected by flow cytometry (FCM). The NK cell behaviors in vitro were analyzed by cell counting kit-8 and cytotoxic activation assays. After incubation with ESCs and macrophages, the expression of CD16, NKG2D, perforin and IFN-γ, viability and cytotoxicity of NK cells were significantly downregulated. The secretion of interleukin (IL)-1ß, IL-10 and transforming growth factor (TGF)-ß in the co-culture system of ESCs and macrophages was increased. Exposure with anti-IL-10 receptor ß neutralizing antibody (αhIL-10Rß) or αTGF-ß could partly reverse these effects of ESCs and macrophages on NK cells in vitro These results suggest that the interaction between macrophages and ESCs downregulates cytotoxicity of NK cells possibly by stimulating the secretion of IL-10 and TGF-ß, and may further trigger the immune escape of ectopic fragments and promote the occurrence and the development of EMS.
Assuntos
Endometriose/metabolismo , Endométrio/metabolismo , Interleucina-10/metabolismo , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Comunicação Parácrina , Células Estromais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adulto , Células Cultivadas , Microambiente Celular , Técnicas de Cocultura , Citotoxicidade Imunológica , Endometriose/imunologia , Endometriose/patologia , Endométrio/imunologia , Endométrio/patologia , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Perforina/metabolismo , Receptores de IgG/metabolismo , Transdução de Sinais , Células Estromais/imunologia , Células Estromais/patologiaRESUMO
During pregnancy, CD8+ T cells are important regulators in the balance of fetal tolerance and antiviral immunity. T-cell immunoglobulin mucin-3 (Tim-3) and programmed cell death-1 (PD-1) are well-recognized negative co-stimulatory molecules involved in viral persistence and tumor metastasis. Here, we demonstrate that CD8+ T cells co-expressing Tim-3 and PD-1 were down-regulated in the deciduae of female mice in abortion-prone matings compared with normal pregnant mice. In addition to their reduced numbers, the Tim-3+PD-1+CD8+ T cells produced lower levels of the anti-inflammatory cytokines interleukin (IL)-4 and IL-10, as well as a higher level of the pro-inflammatory cytokine interferon (IFN)-γ, relative to those from normal pregnancy. Furthermore, normal pregnant CBA/J females challenged with Tim-3- and/or PD-1-blocking antibodies were more susceptible to fetal resorption. These findings indicate that Tim-3 and PD-1 pathways play critical roles in regulating CD8+ T cell function and maintaining normal pregnancy.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Perda do Embrião , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Prenhez/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Animais , Feminino , Tolerância Imunológica , Interferon gama/metabolismo , Masculino , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , GravidezRESUMO
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is frequently altered in human malignancies and Akt over-expression and/or activation induces malignant transformation and chemoresistance. However, the role of Akt in the mechanisms of chemoresistance remains elusive. Here we reported that cisplatin treatment of chemosensitive, but not resistant, ovarian cancer cells (OVCAs) markedly increased the cell proportion in sub-G1 phase. Cisplatin however caused a significant accumulation of the resistant cells in S and G2/M phases, which was associated with a rapid and sustained checkpoint kinase 1 (Chk1) activation. In contrast, while cisplatin also elicited a rapid activation of Chk1 in sensitive cells, it markedly decreased total ChK1 and phospho-Chk1 contents over 12 h. Over-expression of dominant negative (DN)-AKT alone increased phospho-Chk1 content, and induced G2/M arrest and apoptosis. However, it inhibited Chk1 activation and G2/M arrest with combination of cisplatin treatment, resulting in p53-independent apoptosis. Furthermore, the responses of the chemoresistant cells to cisplatin were attenuated with forced expression of constitutive active AKT2. Chk1 knock-down also facilitated cisplatin-induced apoptosis in chemoresistant cells. Our studies implicate that, in addition to its cell survival and anti-apoptotic actions, Akt might also play an important role in the regulation of G2-M transition, possibly via up-regulation of Chk1 activity and stability. These data provide strong support for the concept that Akt is important in cell cycle regulation in the control of chemosensitivity in OVCAs and offers an alternate regulatory pathway for the development of rationale therapy for cisplatin-resistant ovarian cancer.
Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNARESUMO
Physiological pregnancy requires the maternal immune system to recognize and tolerate embryonic Ags. Although multiple mechanisms have been proposed, it is not yet clear how the fetus evades the maternal immune system. In this article, we demonstrate that trophoblast-derived thymic stromal lymphopoietin (TSLP) instructs decidual CD11c(+) dendritic cells (dDCs)with increased costimulatory molecules; MHC class II; and Th2/3-type, but not Th1-type, cytokines. TSLP-activated dDCs induce proliferation and differentiation of decidual CD4(+)CD25(-) T cells into CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) through TGF-ß1. TSLP-activated dDC-induced Tregs display immunosuppressive features and express Th2-type cytokines. In addition, decidual CD4(+)CD25(+)FOXP3(+) Tregs promote invasiveness and HLA-G expression of trophoblasts, resulting in preferential production of Th2 cytokines and reduced cytotoxicity in decidual CD56(bright)CD16(-) NK cells. Of interest, decreased TSLP expression and reduced numbers of Tregs were observed at the maternal-fetal interface during miscarriage. Our study identifies a novel feedback loop between embryo-derived trophoblasts and maternal decidual leukocytes, which induces a tolerogenic immune response to ensure a successful pregnancy.
Assuntos
Citocinas/metabolismo , Células Dendríticas/metabolismo , Histocompatibilidade Materno-Fetal/imunologia , Linfócitos T Reguladores/metabolismo , Aborto Espontâneo/metabolismo , Adulto , Antígeno CD11c/imunologia , Antígenos CD4/metabolismo , Antígeno CD56/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citocinas/imunologia , Decídua/citologia , Decídua/metabolismo , Células Dendríticas/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Antígenos de Histocompatibilidade Classe II , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Gravidez , Receptores de IgG/metabolismo , Células Th2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Trofoblastos/imunologia , Trofoblastos/metabolismo , Adulto Jovem , Linfopoietina do Estroma do TimoRESUMO
STUDY QUESTION: What mechanism is involved in regulating the autophagy of endometrial stromal cells (ESCs), and does it participate in the pathogenesis of endometriosis? SUMMARY ANSWER: CXCL12 down-regulates secretory phase ESC autophagy. WHAT IS KNOWN ALREADY: mTOR (mammalian target of rapamycin), the major negative regulator of autophagy, is abnormally increased in endometriotic lesions and is involved in the direct regulation of endometrial stromal cell (ESC) apoptosis. STUDY DESIGN, SIZE, DURATION: Autophagy was measured by transmission electron microscopy and immunofluorescence, and in vitro analysis was used to measure estrogen/CXCL12/CXCR4 signaling-mediated ESC autophagy. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 31 controls and 31 women with histologically confirmed endometriosis were included. We measured the autophagy level of normal and endometriosis-derived endometrium, and its relationship to the stage of endometriosis, as well as the potential molecular and signaling pathways that mediate the aberrant autophagy in endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with control secretory phase ESCs, a significant reduction of the autophagy grade (as observed in TEM), punctuate LC3B staining (as observed in immunofluorescence assays), and autophagy-associated protein levels were exhibited in secretory phase eutopic ESCs (P < 0.05) and ectopic ESCs (P < 0.05) from women with endometriosis. In addition, the autophagy level was strongly negatively correlated with the CXCL12 concentration in ESCs (R(2) = -0.9694). However, there was no significant difference in autophagy grade or CXCL12 concentration between stage I-II and stage III-IV endometriosis-derived ectopic ESCs (P > 0.05). Based on a human autophagy PCR array, CXCL12 and CXCR4, which is the CXCL12 receptor, in ESCs were predicted to be molecules that mediate the abnormally lower autophagy in endometriosis. Accordingly, after estradiol (E2) treatment a marked increase in CXCL12 secretion (1.71-fold, P < 0.01) and CXCR4 expression (5.07-fold, P < 0.01) in secretory phase ESCs was observed together with decreases in autophagy grade (TEM), punctuate LC3B immunofluorescent staining and autophagy-associated protein levels (P < 0.05). These changes could be reversed by progesterone (P4) (P < 0.05). The suppression of autophagy induced by E2 and recombinant human CXCL12 protein could be abrogated by an anti-CXCR4 neutralizing antibody and by a NF-κB inhibitor (P < 0.05), respectively. In addition, estrogen-stimulated CXCL12 secretion led to a low population of S phase cells (P < 0.05), as well as a low level of apoptosis (P < 0.05) in secretory phase ESCs. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to examine the mechanism of autophagy on ESC apoptosis. WIDER IMPLICATIONS OF THE FINDINGS: Measures to increase in endometrial autophagy might be a valid, novel approach to reduce local E2-dependent growth of endometriotic tissue. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Natural Science Foundation of China (NSFC) (81471513, 81471548 and 81270677), the Training Program for Young Talents of Shanghai Health System XYQ2013104, the Program for Zhuoxue of Fudan University, and the Program for Creative Talents Education of Key Disciplines of Fudan University. None of the authors has any conflict of interest to declare.
Assuntos
Autofagia/fisiologia , Quimiocina CXCL12/metabolismo , Endometriose/fisiopatologia , Endométrio/metabolismo , Estrogênios/fisiologia , Receptores CXCR4/metabolismo , Transdução de Sinais/fisiologia , Células Estromais/fisiologia , Adulto , Endometriose/metabolismo , Endométrio/citologia , Estrogênios/metabolismo , Feminino , Humanos , Células Estromais/metabolismo , Regulação para CimaRESUMO
Chemokine CCL24, acting through receptor CCR3, is a potent chemoattractant for eosinophil in allergic diseases and parasitic infections. We recently reported that CCL24 and CCR3 are co-expressed by trophoblasts in human early pregnant uterus. Here we prove with evidence that steroid hormones estradiol (E), progesterone (P), and human chorionic gonadotropin (hCG), as well as decidual stromal cells (DSCs) could regulate the expression of CCL24 and CCR3 of trophoblasts. We further investigate how trophoblast-derived CCL24 mediates the function of trophoblasts in vitro, and conclude that CCL24/CCR3 promotes the proliferation, viability and invasiveness of trophoblasts. In addition, analysis of the downstream signaling pathways of CCL24/CCR3 show that extracellular signal-regulated kinases (ERK1/2) and phosphoinositide 3-kinase (PI3K) pathways may contribute to the proliferation, viability and invasiveness of trophoblasts by activating intracellular molecules Ki67 and matrix metallopeptidase 9 (MMP9). However, we did not observe any inhibitory effect on trophoblasts when blocking c-Jun N-terminal kinase (JNK) or p38 pathways. In conclusion, our data suggests that trophoblast-derived CCL24 at the maternal-fetal interface promotes trophoblasts cell growth and invasiveness by ERK1/2 and PI3K pathways. Meanwhile, pregnancy-related hormones (P and hCG), as well as DSCs could up-regulate CCL24/CCR3 expression in trophoblasts, which may indirectly influence the biological functions of trophoblasts. Thus, our results provide a possible explanation for the growth and invasion of trophoblasts in human embryo implantation.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL24/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Trofoblastos/patologia , Adulto , Apoptose/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Gravidez , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Adulto JovemRESUMO
Interleukin (IL)-33, a newly described member of the IL-1 family, has been reported to facilitate primary tumor progression and metastatic dissemination. However, its biological function on decidual stromal cells (DSCs) remains unclear. In this study, we tested the hypothesis whether IL-33 promotes proliferation and invasion of DSCs, and the possible mechanism. IL-33 and its orphan receptor ST2 was found to be co-expressed by DSCs in human first-trimester pregnancy. Addition of IL-33, enhanced the proliferation and invasion of DSCs in a dosage-dependent manner, concomitantly with increasing expression of proliferation relative gene (PCNA, survivin) and invasion relative gene (titin, MMP2). Blocking IL-33/ST2 signaling by soluble sST2 apparently abolished the stimulatory effect on the proliferation, invasiveness and related gene expression in DSCs. We also demonstrated that chemokines CCL2/CCR2 was significantly increased with IL-33 administration. Moreover, inhibition of CCL2/CCR2 activation using CCL2 neutralizing antibody or CCR2 blocker prevented IL-33-stimulated proliferation and invasiveness capacity of DSCs. Increasing phosphorylation of nuclear factor NF-κB p65 and extracellular signal-regulated kinases ERK1/2 after treatment with IL-33 was confirmed by western blotting. And the IL-33-induced CCL2/CCR2 expression was abrogated by treatment with the NF-κB inhibitor BAY 11-7082 or ERK1/2 inhibitor U0126. Finally, we showed that decreased IL-33/ST2 expression was observed in DSCs from spontaneous abortion compared with normal pregnancy at both gene and protein levels. This study provides evidence for the molecular mechanism of IL-33 in promoting proliferation and invasiveness of DSCs by up-regulation of CCL2/CCR2 via NF-κB and ERK1/2 signal pathways and thus contributes insight to the potential of IL-33 involved in successful pregnancy via inducing DSCs mitosis and invasion.
Assuntos
Quimiocina CCL2/genética , Decídua/efeitos dos fármacos , Interleucinas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , NF-kappa B/genética , Receptores CCR2/genética , Células Estromais/efeitos dos fármacos , Adulto , Anticorpos/farmacologia , Butadienos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/metabolismo , Decídua/citologia , Decídua/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-33 , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Nitrilas/farmacologia , Gravidez , Primeiro Trimestre da Gravidez , Cultura Primária de Células , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptores CCR2/antagonistas & inibidores , Receptores CCR2/metabolismo , Transdução de Sinais , Células Estromais/citologia , Células Estromais/metabolismo , Sulfonas/farmacologia , SurvivinaRESUMO
Spontaneous abortion is the most common complication of pregnancy. Immune activation and the subsequent inflammation-induced tissue injury are often observed at the maternal-fetal interface as the final pathological assault in recurrent spontaneous abortion. However, the precise mechanisms responsible for spontaneous abortion involving inflammation are not fully understood. Chemokine CCL28 and its receptors CCR3 and CCR10 are important regulators in inflammatory process. Here, we examined the expression of CCL28 and its receptors in decidual stromal cells (DSCs) by immunochemistry and flow cytometry (FCM), and compared their expression level in DSCs from normal pregnancy versus spontaneous abortion, and their relationship to inflammatory cytokines production by DSCs. We further analyzed regulation of the pro-inflammatory cytokines on CCL28 expression in DSCs by real-time polymerase chain reaction, In-cell Western and FCM. The effects of CCL28-CCR3/CCR10 interaction on DSC apoptosis was investigated by Annexin V staining and FCM analysis or DAPI staining and nuclear morphology. Higher levels of the inflammatory cytokines interleukin (IL)-1ß, IL-17A and tumor necrosis factor-α, and increased CCR3/CCR10 expression were observed in DSCs from spontaneous abortion compared with normal pregnancy. Treatment with inflammatory cytokines differently affected CCL28 and CCR3/CCR10 expression in DSCs. Human recombinant CCL28 promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10 and CCL28. However, CCL28 did not affect DSC growth. These results suggest that the inflammation-promoted up-regulation of CCL28 and its receptors interaction in DSCs is involved in human spontaneous abortion via inducing DSC apoptosis.
Assuntos
Aborto Espontâneo/metabolismo , Quimiocinas CC/metabolismo , Decídua/citologia , Receptores CCR10/metabolismo , Receptores CCR3/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Aborto Espontâneo/genética , Adulto , Apoptose/efeitos dos fármacos , Apoptose/genética , Células Cultivadas , Quimiocinas CC/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Técnicas In Vitro , Interleucina-17/farmacologia , Interleucina-1beta/farmacologia , Gravidez , Receptores CCR10/genética , Receptores CCR3/genética , Células Estromais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adulto JovemRESUMO
Interleukin-24 (IL-24) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells. The present study aimed to elucidate the biological function of IL-24 and its receptors (IL-20R1, IL-20R2 and IL-22R1) in decidual stromal cells (DSCs) at human maternal-fetal interface. The DSCs behaviors in vitro were verified by viability (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptosis assay, respectively. Additionally, the effects of pregnancy-associated hormones on IL-24 and the effect of IL-24 on the correspondent functional molecules were investigated by ELISA, in-cell western and flow cytometry, respectively. Here we found that DSCs expressed IL-24 and its receptors, and IL-24 obviously suppressed the viability and stimulated the apoptosis in DSCs. On the contrary, both anti-IL-24 and IL-22R1 neutralizing antibodies markedly promoted growth and reduced the apoptosis. Estrogen but not progesterone could significantly decrease IL-24 but not its receptors, and these effects could be abolished by the antagonist of estrogen receptor beta (ERß). IL-24 significantly restricted the stimulatory effect of estrogen on the viability, anti-apoptosis, anti-apoptosis gene Bcl-2 and proliferation relative gene Ki-67 in DSCs. Our study has demonstrated that IL-24/IL-20R2/IL-22R1 axis is involved in the regulation of estrogen/ERß signaling on the growth of DSCs through up-regulating the expression of Bcl-2 and Ki67, which suggests that estrogen plays an important role in DSC growth of the early pregnancy through down-regulating IL-24.
Assuntos
Estrogênios/farmacologia , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Adulto , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Interleucinas/metabolismo , Gravidez , Adulto JovemRESUMO
STUDY QUESTION: Is Nometastatic gene 23-H1 (NME1, also known as nm23-H1) involved in regulating the biological behavior of endometrial stromal cells (ESCs), and does it participate in the pathogenesis of endometriosis? SUMMARY ANSWER: NME1 suppression induces ESC dysfunction in the endometriotic milieu. WHAT IS KNOWN ALREADY: NME1 is a wide-spectrum tumor metastasis suppressor gene that plays an important role in suppressing the invasion and metastasis of tumor cells. STUDY DESIGN, SIZE, DURATION: An in vitro investigation of the effect of NME1 on the proliferation, adhesion and invasion of eutopic ESCs from patients with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary ESCs were prepared from 12 samples of ectopic endometrial tissue (6 peritoneal and 6 ovarian lesions), 18 samples of eutopic endometrial tissues (16 from women with ovarian and 2 from women with pelvic endometriomas) and 12 samples of normal endometrial tissue from women without endometriosis, after the tissues had been analyzed histologically. The growth, invasiveness and adhesion of ESCs were studied by the 5-bromo-2'-deoxyuridine cell proliferation assay and by the Matrigel invasion and adhesion assay. Additionally, the effects of NME1 on the activation or expression of related regulatory proteins were investigated by in-cell Western and flow cytometry assays. MAIN RESULTS AND THE ROLE OF CHANCE: Expression of NME1 in ESCs derived from eutopic or ectopic endometrium from women with endometriosis is lower than in ESCs from women without endometriosis. Estrogen could down-regulate NME1 expression in ESCs. Silencing NME1 in ESCs promoted the expression of proliferating cell nuclear antigen (PCNA), the anti-apoptotic molecule, survivin, and the adhesion-related molecules, integrin ß1 and integrin ανß3. Silencing NME1 also stimulated ESC proliferation, adhesion and invasion but these effects were inhibited by MAPK/Erk and/or Akt blockers. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to examine the regulatory mechanism of estrogen on NME1 expression of ESCs. WIDER IMPLICATIONS OF THE FINDINGS: Abnormally low expression of NME1 in ESCs may be involved in the pathogenesis of endometriosis by up-regulating growth, adhesion and invasion of ESCs via activating the Akt and MAPK/Erk1/2 signal pathways. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by National Natural Science Foundation of China (NSFC) (31270969, 31101064 and 81270677) and Program for ZhouXue of Fudan University. None of the authors has any conflict of interest to declare.
Assuntos
Endometriose/patologia , Endométrio/patologia , Nucleosídeo NM23 Difosfato Quinases/fisiologia , Proteína Oncogênica v-akt/metabolismo , Adesão Celular , Proliferação de Células , Regulação para Baixo/efeitos dos fármacos , Endometriose/metabolismo , Endométrio/metabolismo , Estrogênios/farmacologia , Feminino , Citometria de Fluxo , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Sistema de Sinalização das MAP Quinases , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , SurvivinaRESUMO
Endometriosis is the most common cause of infertility. Endometrial receptivity has been suggested to contribute to infertility and poor reproductive outcomes in affected women. Even though experimental and clinical data suggest that the endometrium differs in women with endometriosis, the pathogenesis of impaired endometrial receptivity remains incomplete. Therefore, this review summarizes the potential mechanisms that affect endometrial function and contribute to implantation failure. Contemporary data regarding hormone imbalance, inflammation, and immunoregulatory dysfunction will be reviewed here. In addition, genetic, epigenetic, glycosylation, metabolism and microRNA in endometriosis-related infertility/subfertility will be summarized. We provide a brief discussion and perspectives on their future clinical implications in the diagnosis and therapy to improve endometrial function in affected women.