RESUMEN
Lipocalin 2 (LCN2) is an induced stressor that promotes the epithelial-mesenchymal transition (EMT). We previously demonstrated that the development of endometriosis in mice correlates with the secretion of LCN2 in the uterus. Here, we sought to clarify the relationship between LCN2 and EMT in endometrial epithelial cells and to determine whether LCN2 plays a role in endometriosis. Antibodies that functionally inhibit LCN2 slowed the growth of ectopic endometrial tissue in a mouse model of endometriosis, suggesting that LCN2 promotes the formation of endometriotic lesions. Using nutrient deprivation as a stressor, LCN2 expression was induced in cultured primary endometrial epithelial cells. As LCN2 levels increased, the cells transitioned from a round to a spindle-like morphology and dispersed. Immunochemical analyses revealed decreased levels of cytokeratin and increased levels of fibronectin in these endometrial cells, adhesive changes that correlate with induction of cell migration and invasion. Lcn2 knockdown also indicated that LCN2 promotes EMT and migration of endometrial epithelial cells. Our results suggest that stressful cellular microenvironments cause uterine tissues to secrete LCN2 and that this results in EMT of endometrial epithelial cells, which may correlate with the development of ectopic endometriosis. These findings shed light on the role of LCN2 in the pathology of endometrial disorders.
Asunto(s)
Proteínas de Fase Aguda/fisiología , Endometriosis/etiología , Endometrio/citología , Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal/fisiología , Lipocalinas/fisiología , Proteínas Oncogénicas/fisiología , Proteínas de Fase Aguda/inmunología , Proteínas de Fase Aguda/metabolismo , Animales , Anticuerpos/farmacología , Movimiento Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Endometrio/química , Femenino , Fibronectinas/análisis , Queratinas/análisis , Lipocalina 2 , Lipocalinas/inmunología , Lipocalinas/metabolismo , Masculino , Ratones , Proteínas Oncogénicas/inmunología , Proteínas Oncogénicas/metabolismo , Útero/metabolismoRESUMEN
OBJECTIVE: To investigate the location of ß-1,4-N-acetylgalactosaminyltransferase II (B4GALNT2) and the involvement of this protein and Sd(a) antigen in embryonic implantation. DESIGN: Cell and animal study. SETTING: University. ANIMAL(S): Adult outbred Institute for Cancer Research mice. INTERVENTION(S): B4GALNT2 antibody injected into the uteri of mice in early pregnancy; E3.5 blastocysts and pregnant uterine tissues were collected. MAIN OUTCOME MEASURE(S): Protein expression was detected by immunofluorescence staining and Western blotting. Embryo attachment was assayed via in vitro and in vivo embryo implantation models. RESULT(S): The b4galnt2 gene expression in the 293T cell line showed the protein localized in the plasma membrane. We confirmed that B4GALNT2 was localized on the surface of E3.5 blastocysts but was an intracellular component in uterine epithelia. Finally, anti-B4GALNT2 and lectins inhibition assays demonstrated the involvement of B4GALNT2 and Sd(a) antigen in embryonic attachment in vitro and in vivo via the mouse system and human endometrial cell line (Ishikawa). CONCLUSION(S): B4GALNT2 expressed in the blastocyst may interact with a ligand on the endometrial surface, perhaps via Sd(a) also, to permit embryo implantation. Our data suggest that B4GALNT2 and Sd(a) antigen are essential for embryo implantation.
Asunto(s)
Blastocisto/enzimología , Implantación del Embrión , N-Acetilgalactosaminiltransferasas/metabolismo , Útero/enzimología , Animales , Anticuerpos/administración & dosificación , Blastocisto/efectos de los fármacos , Blastocisto/inmunología , Western Blotting , Membrana Celular/enzimología , Membrana Celular/inmunología , Técnicas de Cultivo de Embriones , Endometrio/enzimología , Endometrio/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Edad Gestacional , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Antígenos de Histocompatibilidad/metabolismo , Humanos , Inyecciones , Ligandos , Ratones , N-Acetilgalactosaminiltransferasas/antagonistas & inhibidores , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/inmunología , Embarazo , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Útero/efectos de los fármacos , Útero/inmunologíaRESUMEN
Recently, endometrial hyperplasia was identified as presenting a higher risk for progressing to endometrial carcinoma more readily than adenomyosis. The Lcn-2 gene encodes neutrophil gelatinase-associated lipocalin (NGAL), which promotes cell proliferation and serves as a cancer marker in some cancers. In our current study, we investigated the relationship between the expression of NGAL and that of pathogenic cytokines and cancer-related genes including cyclooxygenase-2 (COX-2), E-cadherin, ß-catenin, and vimentin in patients with endometrial disorders. NGAL expression was examined by Western blotting, immunohistochemistry, and reverse-transcription polymerase chain reaction (RT-PCR) in hyperplasia and adenomyosis biopsy samples. Immunohistochemistry demonstrated the occurrence of NGAL in glandular epithelial cells but not in the stromal cells of hyperplasia biopsy samples. NGAL protein and mRNA expression were significantly greater in endometrial hyperplasia than in endometrial adenomyosis. Although our data showed no difference in pathogenic cytokines between patients with endometrial hyperplasia and endometrial adenomyosis, we observed high expression levels of COX-2, ß-catenin, vimentin, and E-cadherin in patients with endometrial hyperplasia. NGAL mRNA expression correlated positively with COX-2 and E-cadherin mRNA expression (r = 0.41 and r = 0.57, respectively), but correlated negatively with vimentin and ß-catenin mRNA expression (r = -0.42 and r = -0.61, respectively). Our data suggest that NGAL is up-regulated in patients with endometrial hyperplasia to prevent the transition from hyperplasia to carcinoma.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Biomarcadores de Tumor/metabolismo , Hiperplasia Endometrial/metabolismo , Endometriosis/metabolismo , Lipocalinas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Análisis de Varianza , Western Blotting , Cadherinas/metabolismo , Ciclooxigenasa 2/metabolismo , Cartilla de ADN/genética , Células Epiteliales/metabolismo , Femenino , Humanos , Inmunohistoquímica , Lipocalina 2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vimentina/metabolismo , beta Catenina/metabolismoRESUMEN
OBJECTIVE: To investigate B4galnt2 gene regulation in the female mouse reproductive system (B4galnt2 encodes an enzyme, ß1,4-N-acetylgalactosylaminyltransferase II, that catalyzes the addition of GalNAc to glycoproteins via a ß1,4 linkage). DESIGN: Experimental prospective study. SETTING: Research institute and university. ANIMAL(S): Outbred Institute for Cancer Research (ICR) mice. INTERVENTION(S): Subcutaneous injection of P/E2; uterine tissues were collected after a 3-day injection period and were collected at different times during pregnancy. MAIN OUTCOME MEASURE(S): Gene expression was measured by quantitative real-time polymerase chain reaction after hormonal treatment of ovariectomized mice or pregnant mice. Primary endometrial cell cultivation and a gene promoter assay were used for P regulation analysis. The small interfering RNA (siRNA) technique was used to assess the gene function in embryo implantation in vivo. RESULT(S): Animal experiments, a primary endometrial cell cultivation assay, and a gene promoter assay indicated that B4galnt2 is regulated positively by P and negatively by estrogen. B4galnt2 was expressed in uterine tissue at peri-implantation (embryonic day 3.5) along with a sharp increase in placental P production at embryonic day 10.5, and declined as estrogen increased during pregnancy. Using the siRNA in vivo implantation assay, we have proved that B4galnt2 participated in embryonic implantation during pregnancy in mice. CONCLUSION(S): This study shows for the first time the expression of B4galnt2 in pregnant mice and its regulation by P. We conclude that the naturally occurring up-regulation of B4galnt2 during pregnancy contributes to normal embryo implantation but not to embryo development.
Asunto(s)
Implantación del Embrión , N-Acetilgalactosaminiltransferasas/metabolismo , Progesterona/administración & dosificación , Útero/efectos de los fármacos , Análisis de Varianza , Animales , Secuencia de Bases , Células Cultivadas , Implantación del Embrión/genética , Estradiol/administración & dosificación , Femenino , Regulación de la Expresión Génica , Genes Reporteros , Edad Gestacional , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , N-Acetilgalactosaminiltransferasas/genética , Ovariectomía , Reacción en Cadena de la Polimerasa , Embarazo , Regiones Promotoras Genéticas/efectos de los fármacos , Interferencia de ARN , Útero/enzimologíaRESUMEN
Lipocalin-2 (Lcn-2) is an acute-phase protein that has been implicated in diverse physiological processes in mice, including: apoptosis, ion transport, inflammation, cell survival, and tumorigenesis. This study characterized the biological activity of Lcn-2 in human endometrial carcinoma cells (RL95-2). Exposure of RL95-2 cells to Lcn-2 for >24 h reduced Lcn-2-induced cell apoptosis, changed the cell proliferation and up-regulated cytokine secretions, including: interleukin-8 (IL-8), inteleukin-6 (IL-6), monocyte chemotatic protein-1 (MCP-1) and growth-related oncogene (GRO). However, IL-8 mRNA and protein levels were dramatically increased in Lcn-2-treated RL95-2 cells. To determine the IL-8 effect on Lcn-2-treated RL95-2 cells was our major focus. Adding recombinant IL-8 (rIL-8) resulted in decreased caspase-3 activity in Lcn-2-treated cells, whereas the addition of IL-8 antibodies resulted in significantly increased caspase-3 activity and decreased cell migration. Data indicate that IL-8 plays a crucial role in the induction of cell migration. Interestingly, Lcn-2-induced cytokines, secretion from RL95-2 cells, could not show the potent cell migration ability with the exception of IL-8. We conclude that Lcn-2 triggered cytokine secretions to prevent RL95-2 cells from undergoing apoptosis and subsequently increased cell migration. We hypothesize that Lcn-2 increased cytokine secretion by RL95-2 cells, which in turn activated a cellular defense system. This study suggests that Lcn-2 may play a role in the human female reproductive system or in endometrial cancer.
Asunto(s)
Proteínas de Fase Aguda/farmacología , Carcinoma/metabolismo , Citocinas/biosíntesis , Neoplasias Endometriales/metabolismo , Lipocalinas/farmacología , Proteínas Proto-Oncogénicas/farmacología , Proteínas de Fase Aguda/fisiología , Apoptosis , Carcinoma/patología , Caspasa 3/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Medios de Cultivo Condicionados , Neoplasias Endometriales/patología , Femenino , Humanos , Interleucina-8/metabolismo , Interleucina-8/farmacología , Lipocalina 2 , Lipocalinas/fisiología , Proteínas Proto-Oncogénicas/fisiología , ARN Mensajero/metabolismoRESUMEN
Arsenic trioxide (As2O3) has recently received a great deal of attention because of its capacity to cause complete remission of acute promyelocytic leukemia (APL). To evaluate possible toxicity on the male reproductive system during arsenic therapy, male mice were used as a model. Outbred mice (ICR/CD1 and S-W, 6 weeks old) were subcutaneously administered As2O3 continuously for 5 days, with a 2-day interval, for a period of 3 weeks. As2O3 doses were 0, 0.15, 0.3, 1.5, and 3.0 mg/kg of body weight, respectively. No mice died in any dosage group. Our data showed no significant changes in food consumption or in the weight of the body, liver, testis, or epididymis after As2O3 treatment. Using histological observation to identify the stages of seminiferous tubules, we showed that As2O3 treatment resulted in the inhibition of spermatogenesis. The frequency of mature seminiferous tubules (stages VII and VIII) was markedly decreased after As2O3 treatment. A significant decrease in sperm motility and viability also was found with computer-assisted sperm analysis (CASA) and a SYBR14/PI staining assay. Using an enzyme-linked immunosorbent assay (ELISA), we found a significant decrease in levels of plasma luteinizing hormone (LH) at a dose of 3.0 mg/kg body weight. No significant difference was found in plasma follicle-stimulating hormone (FSH) in all dosages. A significant decrease was found in plasma testosterone in all dosages, but no difference in intratesticular testosterone, with the exception of As2O3 at a dose of 3.0 mg/kg body weight. Moreover, there was a significant decrease in the levels of mRNA involved in testicular testosterone synthesis, cytochrome P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), and cytochrome P450 17-alpha hydroxylase/C17-20 lyase (Cyp17). The use of immunohistological observation showed no obvious difference in the testosterone level of Leydig cells of mice treated with As2O3 at doses of 0.3 and 1.5 mg/kg body weight. We concluded that As2O3 treatment caused damage to sperm mobility and viability. As2O3 treatment disturbed spermatogenesis via reducing gene expression of the key enzymes in testosterone synthesis.