RESUMEN
Extracellular lysophosphatidic acid (LPA) and the G-protein-coupled LPA receptors (LPAR) are involved in cell migration and invasion and found in the human endometrium. However, underlying mechanisms resulting in cellular invasion have been rarely investigated. We used stromal endometrial T-HESC, epithelial endometriotic 12Z, 49Z and Ishikawa cells. Interestingly, proliferation of T-HESC cells was strongly increased after LPA treatment, whereas the epithelial cell lines only showed a moderate increase. LPA increased invasion of 12Z and 49Z strongly and significantly. The LPAR inhibitor Ki16425 (LPAR1/3) attenuated significantly LPA-induced invasiveness of 12Z, which was confirmed by LPAR1 and LPAR3 siRNAs, showing that both LPA receptors contribute to invasiveness of 12Z cells. Investigation of cell invasion with an antibody-based protease array revealed mainly differences in cathepsins and especially cathepsin B between 12Z compared to the less invasive Ishikawa. Stimulation with LPA showed a time- and dose-dependent increased secretion of cathepsin B which was inhibited by the Gq inhibitor YM-254890 and Gi/o inhibitor pertussis toxin in the 12Z cells, again highlighting the importance of LPAR1/3. The activity of intracellular and secreted cathepsin B was significantly upregulated in LPA-treated samples. Inhibition of cathepsin B with the specific inhibitor CA074 significantly reduced LPA-increased invasion of 12Z. Our results reveal a novel role of LPA-mediated secretion of cathepsin B which stimulated invasion of endometriotic epithelial cells mainly via LPAR1 and LPAR3. These findings may deepen our understanding how endometriotic cells invade into ectopic sites, and provide new insights into the role of LPA and cathepsin B in cellular invasion.
Asunto(s)
Catepsina B/metabolismo , Endometriosis/metabolismo , Lisofosfolípidos/efectos adversos , Regulación hacia Arriba , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Isoxazoles/farmacología , Propionatos/farmacología , Receptores del Ácido Lisofosfatídico/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: A substantial body of studies supports the view that molecular and cellular features of endometriotic lesions differ from those of eutopic endometrium. Apart from that, evidence exists that the eutopic endometrium from pa-tients with endometriosis differs from that of females without endometriosis. OBJECTIVE: Aberrant expression profiles include a number of non-steroid signaling pathways that exert their putative influ-ence on the pathogenesis of endometriosis at least in part via crosstalk(s) with estrogen-mediated mechanisms. A rational to focus research on non-steroid signal pathways is that they might be remunerative targets for the development and selection of novel therapeutics to treat endometriosis possibly without affecting estrogen levels. RESULTS AND CONCLUSION: In this article, we describe molecular and cellular features of endometriotic lesions and focus on the canonical WNT/ß-signaling pathway, a key regulatory system in biology (including stem cell homeostasis) and often in pathophysiological conditions such as endometriosis. Recently emerged novel biological concepts in signal transduction and gene regulation like exosomes and microRNAs are discussed in their putative role in the pathogenesis of endometriosis.
RESUMEN
The adherens junction associated protein 1 (AJAP1, aka shrew-1) is presumably a type-I transmembrane protein localizing and interacting with the E-cadherin-catenin complex. In various tumors, AJAP1 expression is reduced or lost, including hepatocellular and esophageal squamous cell carcinoma, and glial-derived tumors. The aberrant expression of AJAP1 is associated with alterations in cell migration, invasion, increased tumor growth, and tumor vascularization, suggesting AJAP1 as a putative tumor suppressor. We show that AJAP1 attenuates sprouting angiogenesis by reducing endothelial migration and invasion capacities. Further, we show for the first time that endogenous AJAP1 is associated with the microtubule cytoskeleton. This linkage is independent from cell confluency and stable during angiogenic sprouting in vitro Our work suggests that AJAP1 is a putative negative regulator of angiogenesis, reducing cell migration and invasion by interfering with the microtubule network. Based on our results and those of other authors, we suggest AJAP1 as a novel tumor suppressor and diagnostic marker.
RESUMEN
Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with ß-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.
RESUMEN
In the endometrium transforming growth factor-betas (TGF-ßs) are involved mainly in menstruation and endometriosis. After binding of the ligands to the high-affinity receptors, TGF-ß receptors (TBR1 and TBR2), TGF-ßs activate Smad signaling to modulate gene expression and cellular functions. However, recently also Smad-independent pathways have been studied in more details. To evaluate both pathways, we have analyzed TGF-ß signaling in human endometrial and endometriotic cells. Although endometrial and endometriotic cells secrete TGF-ß1, secretion by stromal cells was higher compared to epithelial cells. In contrast, secretion of TGF-ß2 was higher in endometriotic stromal and endometriotic epithelial cells compared to normal endometrial cells. Treatment of endometrial and endometriotic stromal and epithelial cells with TGF-ß1 or TGF-ß2 increased Smad-dependent secretion of plasminogen activator inhibitor-1 (PAI-1) dramatically in all three cell lines. Of note, endometriotic cells secreted clearly higher levels of PAI-1 compared to endometrial cells. Whereas a TBR1 kinase inhibitor completely blocked the TGF-ß1 or TGF-ß2-induced PAI-1 secretion, an ERK1/2 inhibitor only partially reduced PAI-1 secretion. This inhibition was not dependent on epidermal growth factor receptor (EGFR) activation by phosphorylation but on kinase activity of the TBR1. Finally, treatment of endometrial and endometriotic cell lines with recombinant PAI-1 showed reduced cell adhesion, especially of the endometrial cells. In summary, our results demonstrate that both Smad-dependent and TBR1-dependent ERK1/2 pathways are necessary for TGF-ß-dependent high level secretion of PAI-1, which might increase cellular deadhesion.
RESUMEN
OBJECTIVE: To study the expression and function of syndecan-4 in endometriosis. DESIGN: Histopathological investigation of eutopic endometrium and experimental laboratory study on a cell line derived from epithelial endometriotic cells (12Z). SETTING: University hospital laboratory. PATIENT(S): One hundred six women (62 controls/44 endometriosis) from the IVF center of Münster University Hospital aged 23-44 undergoing Pipelle biopsy and diagnostic exploratory laparoscopy. INTERVENTION(S): Eutopic endometrial tissue was investigated by immunohistochemistry for the expression of syndecan-4. The human endometriotic cell line 12Z was transiently transfected with syndecan-4 small interfering RNA and investigated for changes in cell behavior. MAIN OUTCOME MEASURE(S): Syndecan-4 expression in eutopic endometrium was evaluated immunohistochemically in endometrial glands and stroma. Scoring results were correlated with the stages of the menstrual cycle and presence or absence of endometriosis. Quantitative polymerase chain reaction was used to measure syndecan-4-dependent expression changes of MMP2, MMP3, MMP9, Rac1, and ATF2. Altered cell behavior was monitored by matrigel invasion assays and cell viability assays. RESULT(S): Syndecan-4 expression was significantly higher in the glands and stroma of patients with endometriosis compared with controls, whereas no menstrual cycle-dependent expression was observed. In 12Z cells, syndecan-4 depletion did not affect cell viability but resulted in a significantly reduced matrigel invasiveness and reduced expression of the small GTPase Rac1, the transcription factor ATF-2, and MMP3. CONCLUSION(S): The upregulation of syndecan-4 in the eutopic endometrium of endometriosis patients may facilitate the pathogenetic process by promoting invasive cell growth via Rac1, MMP3, and ATF-2.
Asunto(s)
Movimiento Celular , Endometriosis/metabolismo , Sindecano-4/metabolismo , Factor de Transcripción Activador 2/metabolismo , Adulto , Estudios de Casos y Controles , Línea Celular , Endometriosis/genética , Endometriosis/patología , Femenino , Humanos , Metaloproteinasa 3 de la Matriz/metabolismo , Fenotipo , Interferencia de ARN , Transducción de Señal , Sindecano-4/genética , Transfección , Regulación hacia Arriba , Adulto Joven , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Endometriosis is characterized by growth of endometrial tissue at ectopic locations. Down-regulation of microRNA miR-200b is observed in endometriosis and malignant disease, driving tumour cells towards an invasive state by enhancing epithelial-to-mesenchymal transition (EMT). miR-200b up-regulation may inhibit EMT and invasive growth in endometriosis. To study its functional impact on the immortalized endometriotic cell line 12Z, the stromal cell line ST-T1b, and primary endometriotic stroma cells, a transient transfection approach with microRNA precursors was employed. Expression of bioinformatically predicted targets of miR-200b was analysed by qPCR. The cellular phenotype was monitored by Matrigel invasion assays, digital-holographic video microscopy and flow cytometry. qPCR revealed significant down-regulation of ZEB1 (P < 0.05) and ZEB2 (P < 0.01) and an increase in E-cadherin (P < 0.01). miR-200b overexpression decreased invasiveness (P < 0.0001) and cell motility (P < 0.05). In contrast, cell proliferation (P < 0.0001) and the stemness-associated side population phenotype (P < 0.01) were enhanced following miR-200b transfection. These properties were possibly due to up-regulation of the pluripotency-associated transcription factor KLF4 (P < 0.05) and require attention when considering therapeutic strategies. In conclusion, up-regulation of miR-200b reverts EMT, emerging as a potential therapeutic approach to inhibit endometriotic cell motility and invasiveness.
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Endometriosis/genética , Proteínas de Homeodominio/genética , Factores de Transcripción de Tipo Kruppel/genética , MicroARNs/fisiología , Proteínas Represoras/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Línea Celular , Movimiento Celular/genética , Proliferación Celular/genética , Regulación hacia Abajo , Endometriosis/patología , Femenino , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Células del Estroma/metabolismo , Regulación hacia Arriba , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/fisiologíaRESUMEN
Endometriosis is a debilitating, estrogen-dependent, progesterone-resistant, inflammatory gynecological disease of reproductive age women. Two major clinical symptoms of endometriosis are chronic intolerable pelvic pain and subfertility or infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent recurrence of disease. There is a fundamental need to identify nonestrogen or nonsteroidal targets for the treatment of endometriosis. Peritoneal fluid concentrations of prostaglandin E2 (PGE2) are higher in women with endometriosis, and this increased PGE2 plays important role in survival and growth of endometriosis lesions. The objective of the present study was to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on molecular and cellular aspects of the pathogenesis of endometriosis and associated clinical symptoms. Using human fluorescent endometriotic cell lines and chimeric mouse model as preclinical testing platform, our results, to our knowledge for the first time, indicate that selective inhibition of EP2/EP4: (i) decreases growth and survival of endometriosis lesions; (ii) decreases angiogenesis and innervation of endometriosis lesions; (iii) suppresses proinflammatory state of dorsal root ganglia neurons to decrease pelvic pain; (iv) decreases proinflammatory, estrogen-dominant, and progesterone-resistant molecular environment of the endometrium and endometriosis lesions; and (v) restores endometrial functional receptivity through multiple mechanisms. Our novel findings provide a molecular and preclinical basis to formulate long-term nonestrogen or nonsteroidal therapy for endometriosis.
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Endometriosis/tratamiento farmacológico , Endometriosis/patología , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/uso terapéutico , Caspasa 3/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Endometrio/irrigación sanguínea , Endometrio/patología , Estrógenos/biosíntesis , Femenino , Humanos , Inflamación/patología , Ratones , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Dolor Pélvico/tratamiento farmacológico , Dolor Pélvico/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Progesterona/metabolismo , Transducción de Señal/efectos de los fármacos , Esteroides/uso terapéutico , Xantonas/farmacología , Xantonas/uso terapéuticoRESUMEN
Endometriosis is an inflammatory gynecological disease of reproductive-age women. The prevalence of endometriosis is 5-10% in reproductive-age women. Modern medical treatments are directed to inhibit the action of estrogen in endometriotic cells. However, hormonal therapies targeting estrogen can be prescribed only for a short time because of their undesirable side effects. Recent studies from our laboratory, using human endometriotic epithelial cell line 12Z and stromal cell line 22B derived from red lesion, discovered that selective inhibition of prostaglandin E2 (PGE2) receptors EP2 and EP4 inhibits adhesion, invasion, growth, and survival of 12Z and 22B cells by modulating integrins, MMPs and TIMPs, cell cycle, survival, and intrinsic apoptotic pathways, suggesting multiple epigenetic mechanisms. The novel findings of the present study indicate that selective pharmacological inhibition of EP2 and EP4: (i) decreases expression of DNMT3a, DNMT3b, H3K9me3, H3K27me3, SUV39H1, HP1a, H3K27, EZH2, JMJD2a, HDAC1, HDAC3, MeCP2, CoREST and Sin3A; (ii) increases expression of H3K4me3, H3H9ac, H3K27ac; and (iii) does not modulate the expression of DNMT1, hSET1, LSD1, MBD1, p300, HDAC2, and JMJD3 epigenetic machinery proteins in an epithelial and stromal cell specific manner. In this study, we report for the first time that inhibition of PGE2-EP2/EP4 signaling modulates DNA methylation, H3 histone methylation and acetylation, and epigenetic memory machinery proteins in human endometriotic epithelial cells and stromal cells. Thus, targeting EP2 and EP4 receptors may emerge as long-term nonsteroidal therapy for treatment of active endometriotic lesions in women.
Asunto(s)
Compuestos de Bifenilo/farmacología , Metilación de ADN/efectos de los fármacos , Endometriosis/genética , Histonas/metabolismo , Antagonistas de Prostaglandina/farmacología , Xantonas/farmacología , Acetilación/efectos de los fármacos , Línea Celular , Endometriosis/tratamiento farmacológico , Endometriosis/metabolismo , Epigénesis Genética/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Endometriosis is characterized by the presence of functional endometrial tissue outside of the uterine cavity. It affects 1 in 10 women of reproductive age. This chronic condition commonly leads to consequences such as pelvic pain, dysmenorrhea, infertility and an elevated risk of epithelial ovarian cancer. Despite the prevalence of endometriosis and its impact on women's lives, there are relatively few in vitro and in vivo models available for studying the complex disease biology, pathophysiology, and for use in the preclinical development of novel therapies. The goal of this study was to develop a novel three-dimensional (3D) cell culture model of ovarian endometriosis and to test whether it is more reflective of endometriosis biology than traditional two dimensional (2D) monolayer cultures. METHODS: A novel ovarian endometriosis epithelial cell line (EEC16) was isolated from a 34-year old female with severe endometriosis. After characterization of cells using in vitro assays, western blotting and RNA-sequencing, this cell line and a second, already well characterized endometriosis cell line, EEC12Z, were established as in vitro 3D spheroid models. We compared biological features of 3D spheroids to 2D cultures and human endometriosis lesions using immunohistochemistry and real-time semi-quantitative PCR. RESULTS: In comparison to normal ovarian epithelial cells, EEC16 displayed features of neoplastic transformation in in vitro assays. When cultured in 3D, EEC16 and EEC12Z showed differential expression of endometriosis-associated genes compared to 2D monolayer cultures, and more closely mimicked the molecular and histological features of human endometriosis lesions. CONCLUSIONS: To our knowledge, this represents the first report of an in vitro spheroid model of endometriosis. 3D endometriosis models represent valuable experimental tools for studying EEC biology and the development of novel therapeutic approaches.
Asunto(s)
Endometriosis/patología , Células Epiteliales/patología , Enfermedades del Ovario/patología , Adulto , Biomarcadores/metabolismo , Línea Celular , Proliferación Celular , Endometriosis/genética , Endometriosis/metabolismo , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Genotipo , Humanos , Enfermedades del Ovario/genética , Enfermedades del Ovario/metabolismo , Fenotipo , Índice de Severidad de la Enfermedad , Esferoides Celulares , Factores de TiempoRESUMEN
OBJECTIVE: To study the function of miR-145, known to be dysregulated in endometriosis, and to identify its target genes in an in vitro endometriosis model. DESIGN: Experimental laboratory study. SETTING: University medical centers. PATIENT(S): Primary endometrial stroma cells were derived from eutopic endometrium of three American Society for Reproductive Medicine stage III endometriosis patients and from ectopic lesions of four patients with deep infiltrating endometriosis. INTERVENTION(S): The human endometriotic cell line 12Z and primary eutopic and ectopic endometrial stroma cells were transiently transfected with miR-145 precursors or anti-miR-145 inhibitors and investigated for posttranscriptional regulation of predicted target genes and changes in cell behavior. MAIN OUTCOME MEASURE(S): Predicted target expression was measured by quantitative reverse transcription-polymerase chain reaction and Western blotting, and altered cell behavior was monitored by cell proliferation assays. The 12Z cells were additionally investigated by Matrigel invasion assays, cell cycle analysis, side population analysis, and aldehyde dehydrogenase activity assays. RESULT(S): In all cells investigated, miR-145 overexpression inhibited cell proliferation and induced down-regulation of FASCIN-1, SOX2, and MSI2. In 12Z cells miR-145 upregulation increased Matrigel invasiveness and reduced side population and aldehyde dehydrogenase-1 activity. Additional down-regulated targets in 12Z cells included OCT4, KLF4, PODXL, JAM-A, and SERPINE1/PAI-1. ACTG2 and TAGLN were up-regulated upon pre-miR-145 transfection. JAM-A, FASCIN-1, and PAI-I down-regulation in 12Z cells were confirmed by Western blotting. CONCLUSION(S): miR-145 inhibits endometriotic cell proliferation, invasiveness, and stemness by targeting multiple pluripotency factors, cytoskeletal elements, and protease inhibitors.
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Citoesqueleto/fisiología , Endometriosis/genética , Endometriosis/patología , MicroARNs/genética , Células Madre Pluripotentes/citología , Adulto , Proteínas Portadoras/genética , Moléculas de Adhesión Celular/genética , Línea Celular , Proliferación Celular , Femenino , Humanos , Técnicas In Vitro , Factor 4 Similar a Kruppel , Proteínas de Microfilamentos/genética , Fenotipo , Inhibidor 1 de Activador Plasminogénico/genética , Células Madre Pluripotentes/fisiología , Procesamiento Postranscripcional del ARN/fisiología , Proteínas de Unión al ARN/genética , Receptores de Superficie Celular/genética , Factores de Transcripción SOXB1/genética , Índice de Severidad de la Enfermedad , Células del Estroma/citología , Células del Estroma/fisiologíaRESUMEN
OBJECTIVE: To study the function of syndecan-1 (SDC1) and its potential regulator miR-10b in endometriosis. DESIGN: Experimental laboratory study. SETTING: University medical center. PATIENT(S): Not applicable. INTERVENTION(S): The human endometriotic cell line 12Z was transiently transfected with SDC1 small interfering RNA or miR-10b precursors and investigated for changes in cell behavior and gene expression. 12Z and primary eutopic endometrial stroma cells of two American Society for Reproductive Medicine class III endometriosis patients were transfected with miR-10b precursors to investigate posttranscriptional regulation of SDC1. MAIN OUTCOME MEASURE(S): Quantitative polymerase chain reaction, Western blotting, flow cytometry, 3' untranslated region luciferase assays, and zymography were used to measure miR-10b-dependent targeting of SDC1 and SDC1-dependent expression changes of proteases and interleukin-6. Altered cell behavior was monitored by Matrigel invasion assays, cell viability assays, and mitogen-activated protein kinase activation blots. RESULT(S): Knockdown of SDC1 inhibited Matrigel invasiveness by >60% but did not affect cell viability. Interleukin-6 secretion, matrix metalloproteinase-9 expression, and matrix metalloproteinase-2 activity were reduced, whereas plasminogen activator inhibitor-1 protein expression was up-regulated. miR-10b overexpression significantly down-regulated SDC1, reduced Matrigel invasiveness by 20% and cell viability by 14%, and decreased mitogen-activated protein kinase activation in response to hepatocyte growth factor. CONCLUSION(S): Syndecan-1, a target of miR-10b, inhibits epithelial endometriotic cell invasiveness through down-regulation of metalloproteinase activity and interleukin-6.
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Endometriosis/genética , Endometrio/citología , Interleucina-6/metabolismo , MicroARNs/genética , Sindecano-1/genética , Materiales Biocompatibles , Línea Celular , Supervivencia Celular/fisiología , Colágeno , Combinación de Medicamentos , Endometriosis/metabolismo , Endometriosis/patología , Femenino , Humanos , Laminina , Sistema de Señalización de MAP Quinasas/fisiología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteoglicanos , ARN Interferente Pequeño/genética , Sindecano-1/metabolismo , TransfecciónRESUMEN
Endometriosis is a chronic gynecological disease of reproductive age women characterized by the presence of functional endometrial tissues outside the uterine cavity. Interactions between the endometriotic cells and the peritoneal extracellular matrix proteins (ECM) are crucial mechanisms that allow adhesion of the endometriotic cells into peritoneal mesothelia. Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. In previous studies, we have reported that selective inhibition of PGE2 receptors PTGER2 and PTGER4 decreases survival and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms. Results of the present study indicates that selective inhibition of PTGER2- and PTGER4-mediated PGE2 signaling 1) decreases the expression and/or activity of specific integrin receptor subunits Itgb1 (beta1) and Itgb3 (beta3) but not Itgb5 (beta5), Itga1 (alpha1), Itga2 (alpha2), Itga5 (alpha5), and Itgav (alphav); 2) decreases integrin-signaling components focal adhesion kinase or protein kinase 2 (PTK2) and talin proteins; 3) inhibits interactions between Itgb1/Itgb3 subunits, PTK2, and talin and PTGER2/PTGER4 proteins through beta-arrestin-1 and Src kinase protein complex in human endometriotic epithelial cells 12Z and stromal cells 22B; and 4) decreases adhesion of 12Z and 22B cells to ECM collagen I, collagen IV, fibronectin, and vitronectin in a substrate-specific manner. These novel findings provide an important molecular framework for further evaluation of selective inhibition of PTGER2 and PTGER4 as potential nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women.
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Endometriosis/etiología , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Arrestinas/metabolismo , Adhesión Celular , Línea Celular , Dinoprostona/metabolismo , Endometriosis/metabolismo , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Talina/metabolismo , beta-Arrestina 1 , beta-Arrestinas , Familia-src Quinasas/metabolismoRESUMEN
Cathepsin B has been shown to be important in angiogenesis; therefore, understanding its regulation in endothelial cells should provide fundamental information that will aid in the development of new treatment options. Peroxisome proliferator-activated receptors (PPARs) have been shown to have anti-inflammatory, anti-angiogenic and anti-tumorigenic properties. We explored the influence of a PPARα agonist on cathepsin B expression in human endothelial cells. The PPARα agonist, Wy14643, was found to inhibit cathepsin B protein expression. Further studies demonstrated the Wy14643-dependent but PPARα-independent suppression of cathepsin B. This has been previously described for other PPAR agonists. Wy14643 suppressed the accumulation of cathepsin B mRNA, which was accompanied by the selective suppression of a 5'-alternative splice variant. Consistent with these results, luciferase promoter assays and electrophoretic mobility shift analysis demonstrated that the suppression was facilitated by reduced binding of the transcription factors USF1/2 to an E-box within the cathepsin B promoter. Additionally, Wy14643 treatment resulted in a reduction in cathepsin B half-life, suggesting a posttranslational regulatory mechanism. Overall, our results suggest that the PPARα-dependent anti-angiogenic action of Wy14643 seems to be mediated, in part, by Wy14643-dependent but PPARα-independent regulation of cathepsin B expression.
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Catepsina B/genética , Catepsina B/metabolismo , Células Endoteliales de la Vena Umbilical Humana/enzimología , PPAR alfa/agonistas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Pirimidinas/farmacología , Transcripción Genética/efectos de los fármacos , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Emparejamiento Base/genética , Western Blotting , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Elementos E-Box/genética , Exones/genética , Semivida , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , PPAR alfa/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Procesamiento Proteico-Postraduccional/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , Factores Estimuladores hacia 5'/metabolismoRESUMEN
Peroxisome proliferator-activated receptor (PPAR) delta agonists are known to have distinct anti-inflammatory and antitumor effects; though, the knowledge regarding their mode of action has thus far been limited. Different cathepsins have been shown to be upregulated in a broad range of pathological events, such as rheumatoid arthritis, psoriasis, atherosclerosis and diverse tumor entities, for example melanoma. Recent work demonstrated that cathepsin B in particular is an important pro-angiogenic protease in various pathological conditions. We therefore analysed whether cathepsins are a valid target for PPARδ agonists. This study reveals an inhibitory effect of two commonly used PPARδ agonists, GW501516 and L-165,041, on the protein expression and enzyme activity of cathepsin B in human endothelial cells. In contrast, no inhibitory effects were observed on cathepsin L and cathepsin D protein expression after treatment with PPARδ agonists. Furthermore, the results substantiate that PPARδ activators mediate their inhibitory action in a PPARδ-dependent manner and that the underlying regulatory mechanism is not based on a transcriptional but rather on a posttranslational mode of action, via the reduction in the cathepsin B protein half-life. Mechanisms conveying the suppressive effect by 5'-alternative splicing, a 3'-UTR-dependent way or by miRNA could be excluded. The data of this study explore cathepsin B as a new valid target for PPARδ agonists in endothelial cells. The results bolster other studies demonstrating PPARδ agonists as anti-inflammatory and anticarcinogenic agents and thus might have the potential to help to develop new pharmaceutical drugs.
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Catepsina B/antagonistas & inhibidores , PPAR delta/agonistas , Regiones no Traducidas 3' , Secuencia de Bases , Catepsina B/genética , Catepsina B/metabolismo , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ligandos , MicroARNs/genética , MicroARNs/metabolismo , PPAR delta/metabolismo , Fenoxiacetatos/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Interferente Pequeño/genética , Ribonucleasa III/antagonistas & inhibidores , Ribonucleasa III/genética , Tiazoles/farmacologíaRESUMEN
Prostaglandin E2 (PGE2) plays an important role in the pathogenesis of endometriosis. We recently reported that inhibition of COX-2 decreased migration as well as invasion of human endometriotic epithelial and stromal cells. Results of the present study indicates that selective inhibition of PGE2 receptors EP2 and EP4 suppresses expression and/or activity of MMP1, MMP2, MMP3, MMP7 and MMP9 proteins and increases expression of TIMP1, TIMP2, TIMP3, and TIMP4 proteins and thereby decreases migration and invasion of human immortalized endometriotic epithelial and stromal cells into matrigel. The interactions between EP2/EP4 and MMPs are mediated through Src and ß-arrestin 1 protein complex involving MT1-MMP and EMMPRIN in human endometriotic cells. These novel findings provide an important molecular and cellular framework for further evaluation of selective inhibition of EP2 and EP4 as potential nonsteroidal therapy for endometriosis in childbearing-age women.
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Endometrio/citología , Células Epiteliales/patología , Inhibidores de la Metaloproteinasa de la Matriz , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Células del Estroma/patología , Movimiento Celular/fisiología , Células Cultivadas , Dinoprostona/metabolismo , Endometriosis/metabolismo , Endometriosis/patología , Células Epiteliales/metabolismo , Femenino , Humanos , ARN Interferente Pequeño/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Células del Estroma/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismoRESUMEN
Targeting of proteins to the endoplasmic reticulum (ER) usually requires N-terminal signal peptides (SP) of approximately 22 amino acids in length. However, a substantial number of proteins contain exceptionally long SPs of 40 amino acids and more, an example being protein shrew-1/AJAP1. Using shrew-1's SP as example, the NtraC model has been developed by dissecting long SPs into two functionally distinct subdomains ("N" and "C") separated by a ß-turn rich transition area ("tra"). Further proteins have been identified by computational analysis complying with the NtraC model. Here we used the SPs of two of these proteins, DCBLD2 and RGMa (including three isoforms), to show that the NtraC model applies to a growing group of SPs. We demonstrate that the full-length SPs of RGMa and DCBLD2 are functional and furthermore that the C-domains are sufficient and essential for ER targeting, whereas the N-domains are dispensable. Thus, the N-domains are available for additional functions.
Asunto(s)
Simulación por Computador , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Algoritmos , Secuencia de Aminoácidos , Células Cultivadas , Biología Computacional , Retículo Endoplásmico/metabolismo , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
OBJECTIVE: To determine interactions between prostaglandin (PG) E(2) signaling and proliferation of endometriotic cells to increase our knowledge about PGE(2) signaling in the pathogenesis of endometriosis in humans. DESIGN: Immortalized human endometriotic epithelial and stromal cells were used as an in vitro model. Effects of inhibition of PGE(2) receptors on proliferation of endometriotic cells and associated cell cycle regulation were determined. SETTING: College Veterinary Medicine and Biomedical Sciences, Texas A&M University. PATIENT(S): Not available. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cell proliferation, cell viability, cell cycle, regulation of cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors. RESULT(S): Selective blockade of EP2/EP4 inhibited proliferation of human endometriotic cells by inducing cell cycle arrest at the G(1)-S and G(2)-M checkpoints in epithelial cells and at the G(2)-M checkpoint in stromal cells. This cell cycle arrest during specific checkpoints was associated with distinct regulation of respective cyclins and cyclin-dependent kinases. Inhibition of EP1 did not decrease endometriotic cell proliferation. CONCLUSION(S): For the first time data from the present study provide a direct molecular link between PGE(2) signaling and proliferation of endometriotic cells and suggest that inhibition of EP2/EP4 could be a potential nonestrogen (E) treatment option for endometriosis in women.
Asunto(s)
Endometriosis/metabolismo , Receptores de Prostaglandina E/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Endometriosis/tratamiento farmacológico , Endometrio/citología , Endometrio/efectos de los fármacos , Femenino , Humanos , Antagonistas de Prostaglandina/farmacología , ARN Interferente Pequeño/farmacología , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Células del Estroma/metabolismo , Xantonas/farmacologíaRESUMEN
The use of anti-L1-cell adhesion molecule monoclonal antibodies (anti-L1CAM-mAb) in an endometriosis epithelial cell line Z12 led to a statistically significant decrease in cell proliferation and cell invasion and to an inhibition of the adhesion compared with unspecific IgG-Ab treated and untreated cells. Because it increases the cell invasion and adhesion which consequently aggravates the disease, L1 could possibly promote endometriosis development; thus, further studies should evaluate the possible use of anti-L1-mAb in an animal endometriosis model.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Endometriosis/patología , Molécula L1 de Adhesión de Célula Nerviosa/inmunología , Enfermedades Peritoneales/patología , Adhesión Celular/efectos de los fármacos , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Modelos Biológicos , Molécula L1 de Adhesión de Célula Nerviosa/antagonistas & inhibidoresRESUMEN
OBJECTIVE(S): To determine whether nuclear factor-kappaB (NF-kappaB) is constitutively and tumor necrosis factor (TNF)-dependently activated in endometriotic cells, whether trichostatin A (TSA) can suppress NF-kappaB activation and suppress TRAF2/6 and TAK1, and whether TSA and caffeic acid phenyl ester can suppress constitutive and H(2)O(2)-stimulated proliferation of endometriotic cells. METHODS: Two endometriotic cell lines and an endometrial stromal cell line were used as an in vitro model. Electrophoretic mobility shift analysis was used to determine NF-kappaB activation and possible suppression by TSA. Western blot analysis was used to determine whether TSA suppresses phosphorylation of IkappaBalpha, phosphorylation of p65 in the cytoplasm and nuclear translocation, and the expression of TRAF2/6 and TAK1. RESULTS: NF-kappaB was constitutively activated in endometriotic cells, but only minimally in endometrial cells. TNFalpha stimulation activated NF-kappaB through induction of IkappaB phosphorylation, but the activation can be suppressed by TSA. TSA also attenuated constitutive and TNF-dependent p65 phosphorylation and nuclear translocation in endometriotic cells. TRAF2, TRAF6 and TAK1 were constitutively activated and were unaffected by TSA treatment. CONCLUSIONS: NF-kappaB activation may play a critical role in the pathogenesis in endometriosis. Targeting NF-kappaB with histone deacetylase inhibitors or other compounds might hold promise as novel therapeutics for endometriosis.