RESUMEN
BACKGROUND: Preeclampsia is a hypertensive disorder of pregnancy characterized by chronic placental ischemia and suppression of proangiogenic proteins, causing oxidative stress, hypertension, and maternal systemic organ damage. The transcription factor, PPARγ (peroxisome proliferator-activated receptor-γ) promotes healthy trophoblast differentiation but is dysregulated in the preeclampsia placenta. Our study identifies the beneficial impact of Rosiglitazone-mediated PPARγ-activation in the stressed preeclampsia placenta. METHODS: We used first trimester placentas, preeclamptic and preterm control placentas, and human trophoblast cell lines to study PPARγ activation. RESULTS: Induction of PPARγ activates cell growth and antioxidative stress pathways, including the gene, heme oxygenase 1 (Hmox1). Protein expression of both PPARγ and HO1 (heme oxygenase 1) are reduced in preeclamptic placentas, but Rosiglitazone restores HO1 signaling in a PPARγ-dependent manner. CONCLUSIONS: Restoring disrupted pathways by PPARγ in preeclampsia offers a potential therapeutic pathway to reverse placental damage, extending pregnancy duration, and reduce maternal sequelae. Future research should aim to understand the full scope of impaired PPARγ signaling in the human placenta and focus on compounds for safe use during pregnancy to prevent severe perinatal morbidity and mortality.
Asunto(s)
Hemo-Oxigenasa 1 , Placenta , Preeclampsia , Femenino , Humanos , Recién Nacido , Embarazo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Placenta/metabolismo , PPAR gamma/efectos de los fármacos , PPAR gamma/metabolismo , Preeclampsia/metabolismo , Rosiglitazona/farmacología , Trofoblastos/metabolismoRESUMEN
Decorin (DCN) is a leucine-rich proteoglycan produced by chorionic villus mesenchymal cells anddecidual cells during human pregnancy. Studies from our laboratory demonstrated that decidua-derived DCN restrains multiple trophoblast functions including proliferation, migration, invasion andendovascular differentiation, mediated by DCN-binding to multiple tyrosine kinase receptors; expressed by the trophoblast. Furthermore, DCN was shown to be selectively over-produced by thedecidua in preeclampsia (PE) subjects and elevated in the second trimester maternal plasma in PE, before the appearance of clinical signs, presenting as a predictive biomarker for PE. Micro (mi)RNAs are single-stranded non-coding RNAs (17-25 nucleotides) that typically downregulate target genes by repressing translation or facilitating degradation of mRNAs. The human; placenta expresses many miRNAs, some of which are exclusively expressed by the trophoblast. Many; of these miRNAs are dysregulated in PE-associated placentas and some appear in the maternal blood as PE biomarkers. However, little is known about their contribution to the pathogenesis of PE, a multi-factorial disease associated with a hypo-invasive placenta. The objective of the present study was to examine whether exposure of extravillous trophoblast (EVT) to DCN affects expression of specific miRNAs, and to test the role of these miRNAs in altering EVT functions. We identified miR-512-3p, as one of the DCN-induced miRNAs, also upregulated in PE placentas. It was shown to be elevated in ectopic DCN-over-expressing or exogenous DCN-treated first trimester human trophoblast cell line HTR-8/SVneo. Use of miRNA-mimics and inhibitors revealed that miR-512-3p compromised trophoblast migration, invasion and VEGF-dependent endovascular differentiation. Finally, Protein Phosphatase 3 Regulatory Subunit B, Alpha (PPP3R1), a known target of miR-512-3p, was paradoxically elevated in miR-512-3p-overexpressing trophoblast and PE-associated placentas. Using Enrichr, a tool that consists of both a validated user-submitted gene list and a search engine for transcription factors, we found that PPP3R1 elevation resulted from the miRNA binding to and targeting Upstream Transcription Factor 2 (USF2) which targeted PPP3R1. These findings reveal a novel aspect of pathogenesis of PE and biomarker potentials of this miRNA in PE.
RESUMEN
Insulin-like growth factor-1 (IGF-1) bioavailability in pregnancy is governed by IGF binding protein (IGFBP-1) and its phosphorylation, which enhances the affinity of IGFBP-1 for the growth factor. The decidua is the predominant source of maternal IGFBP-1; however, the mechanisms regulating decidual IGFBP-1 secretion/phosphorylation are poorly understood. Using decidualized primary human endometrial stromal cells (HESCs) from first-trimester placenta, we tested the hypothesis that mTORC1 signaling mechanistically links hypoxia to decidual IGFBP-1 secretion/phosphorylation. Hypoxia inhibited mechanistic target of rapamycin (mTORC1) (p-P70-S6K/Thr389, -47%, p = 0.038; p-4E-BP1/Thr70, -55%, p = 0.012) and increased IGFBP-1 (total, +35%, p = 0.005; phosphorylated, Ser101/+82%, p = 0.018; Ser119/+88%, p = 0.039; Ser 169/+157%, p = 0.019). Targeted parallel reaction monitoring-mass spectrometry (PRM-MS) additionally demonstrated markedly increased dual IGFBP-1 phosphorylation (pSer98+Ser101; pSer169+Ser174) in hypoxia. IGFBP-1 hyperphosphorylation inhibited IGF-1 receptor autophosphorylation/ Tyr1135 (-29%, p = 0.002). Furthermore, silencing of tuberous sclerosis complex 2 (TSC2) activated mTORC1 (p-P70-S6K/Thr389, +68%, p = 0.038; p-4E-BP1/Thr70, +30%, p = 0.002) and reduced total/site-specific IGFBP-1 phosphorylation. Importantly, TSC2 siRNA prevented inhibition of mTORC1 and the increase in secretion/site-specific IGFBP-1 phosphorylation in hypoxia. PRM-MS indicated concomitant changes in protein kinase autophosphorylation (CK2/Tyr182; PKC/Thr497; PKC/Ser657). Overall, mTORC1 signaling mechanistically links hypoxia to IGFBP-1 secretion/phosphorylation in primary HESC, implicating decidual mTORC1 inhibition as a novel mechanism linking uteroplacental hypoxia to fetal growth restriction.
Asunto(s)
Decidua/patología , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Transducción de Señal , Adulto , Quinasa de la Caseína II/metabolismo , Hipoxia de la Célula , Forma de la Célula , Células Cultivadas , Femenino , Silenciador del Gen , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fosforilación , Embarazo , Primer Trimestre del Embarazo/fisiología , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/metabolismo , Receptor IGF Tipo 1/metabolismo , Células del Estroma/metabolismo , Células del Estroma/patología , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Adulto JovenRESUMEN
Placenta in certain species including the human has evolved as a highly invasive tumor-like organ invading the uterus aned its vasculature to derive oxygen and nutrients for the fetus and exchange waste products. While several excellent reviews have been written comparing hemochorial placentation with tumors, no comprehensive review is available dealing with mechanistic insights into what makes them different, and what tumor biologists can learn from placental biologists, and vice versa. In this review, we analyze the structure-function relationship of the human placenta, emphasizing the functional need of the spatio-temporally orchestrated trophoblast invasiveness for fetal development and growth, and pathological consequences of aberrant invasiveness for fetal and maternal health. We then analyze similarities and differences between the placenta and invasive tumors in terms of hallmarks of cancer, some key molecules regulating their invasive functions, and how placental cancers (choriocarcinomas) or other cancers become refractory or even addicted to these invasion-restraining molecules. We cite in vitro models of human trophoblast and choriocarcinoma cell lines utilized to study mechanisms in normal placental development as well as those responsible for tumor progression. We discuss the pathobiology of hyper-invasive placentas and show thattrophoblastic neoplasias are a unique and heterogeneous class of tumors. We delve into the questions as to why metastasis from other organs rarely occurs at the placental site and whether pregnancy makes the mother more or less vulnerable to cancer-related morbidity/mortality. We attempt to compare trophoblast stem cells and cancer stem cells. Finally, we leave the readers with some thoughts as foods of future investigations.
Asunto(s)
Neoplasias/patología , Placenta/patología , Placentación/fisiología , Coriocarcinoma/patología , Femenino , Humanos , Embarazo , Neoplasias Uterinas/patologíaRESUMEN
Decidual mechanistic target of rapamycin (mTOR) is inhibited, amino acid response (AAR) and protein kinase CK2 are activated, and IGF (insulin-like growth factor) binding protein (IGFBP)-1 is hyperphosphorylated in human intrauterine growth restriction (IUGR). Using decidualized human immortalized endometrial stromal cells (HIESC), we hypothesized that hypoxia and leucine deprivation causing inhibition of decidual IGF-1 signaling is mediated by mTOR, AAR, CK2 and IGFBP-1 phosphorylation. Mass spectrometry demonstrated that hypoxia (1% O2) or rapamycin increased IGFBP-1 phosphorylation singly at Ser101/119/169 (confirmed using immunoblotting) and dually at pSer169 + 174. Hypoxia resulted in mTOR inhibition, AAR and CK2 activation, and decreased IGF-1 bioactivity, with no additional changes with rapamycin + hypoxia. Rapamycin and/or hypoxia promoted colocalization of IGFBP-1 and CK2 (dual-immunofluorescence and proximity ligation assay). Leucine deprivation showed similar outcomes. Changes in IGFBP-1 phosphorylation regulated by mTOR/AAR signaling and CK2 may represent a novel mechanism linking oxygen and nutrient availability to IGF-1 signaling in the decidua.
Asunto(s)
Hipoxia de la Célula/fisiología , Decidua/metabolismo , Leucina/deficiencia , Sistemas de Transporte de Aminoácidos/metabolismo , Quinasa de la Caseína II/metabolismo , Células Cultivadas , Decidua/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Leucina/farmacología , Fosforilación , Receptores de Aminoácidos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Lymphangiogenesis (formation of new lymphatic vessels), unlike angiogenesis, has been a lesser-focused field in cancer biology, because of earlier controversy regarding whether lymphatic metastasis occurs via pre-existing or newly formed lymphatics. Recent evidence reveals that peri-tumoral or intra-tumoral lymphangiogenesis is a precursor for lymphatic metastasis in most carcinomas and melanomas. Two major lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and VEGF-D, are produced by cancer cells or immune cells such as macrophages in the tumor-stroma to promote sprouting of lymphatics from lymphatic endothelial cells (LEC) or LEC precursors (LECP) by binding to their primary (high affinity) receptor VEGF-R3 or secondary receptors VEGF-R2, neuropilin (NRP)2 and α9/ß1 integrin. Many other growth factors/receptors such as VEGF-A/VEGF-R2, fibroblast growth factor (FGF)2/FGF-R, platelet-derived growth factor (PDGF)/PDGF-R, hepatocyte growth factor (HGF)/C-Met, angiopoietins (Ang)1, 2/Tie2, and chemokines/ chemokine receptors (CCL21/CCR7, CCL12/CCR4) can also stimulate LEC sprouting directly or indirectly. This review deals with the roles of prostaglandins (PG), in particular PGE2, in cancer-associated lymphangiogenesis, with special emphasis on breast cancer. We show that cyclooxygenase (COX)-2 expression by breast cancer cells or tumor stroma leading to high PGE2 levels in the tumor milieu promotes lymphangiogenesis and lymphatic metastases, resulting from binding of PGE2 to PGE receptors (EP, in particular EP4) on multiple cell types: tumor cells, tumor-infiltrating immune cells, and LEC. EP4 activation on cancer cells and macrophages upregulated VEGF-C/D production to stimulate LEC sprouting. Furthermore, ligation of EP4 with PGE2 on cancer or host cells can initiate a new cascade of molecular events leading to cross-talk between cancer cells and LEC, facilitating lymphangiogenesis and lympho-vascular transport of cancer cells. We make a case for EP4 as a potential therapeutic target for breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Linfangiogénesis , Neovascularización Patológica , Prostaglandinas/metabolismo , Microambiente Tumoral , Animales , Biomarcadores , Neoplasias de la Mama/complicaciones , Ciclooxigenasa 2/metabolismo , Progresión de la Enfermedad , Eicosanoides/metabolismo , Células Endoteliales/metabolismo , Femenino , Humanos , Técnicas In Vitro , Metástasis Linfática , Linfedema/etiología , Redes y Vías Metabólicas , Receptores de Prostaglandina/metabolismo , Transducción de SeñalRESUMEN
The origin and regulation of stem cells sustaining trophoblast renewal in the human placenta remain unclear. Decorin, a leucine-rich proteoglycan restrains trophoblast proliferation, migration/invasiveness and endovascular differentiation, and local decorin overproduction is associated with preeclampsia (PE). Here, we tested the role of decorin in human trophoblast stem cell self-renewal and differentiation, using two models: an immortalized first trimester trophoblast cell line HTR-8/SVneo (HTR) and freshly isolated primary trophoblast (p-trophoblast) from early first trimester (6-9 weeks) placentas. Self-renewal capacity was measured by spheroid forming ability of single cells on ultra-low attachment plates for multiple generations. Markers of embryonic stem (ES) cells, trophoblast stem (TS) cells and trophoblast were used to identify stem cell hierarchy. Differentiation markers for syncytial and extravillous (EVT) pathways were employed to identify differentiated cells. Bewo cells were additionally used to explore DCN effects on syncytialization. Results reveal that the incidence of spheroid forming stem-like cells was 13-15% in HTR and 0.1-0.4%, in early first trimester p-trophoblast, including a stem cell hierarchy of two populations of ES and TS-like cells. DCN restrained ES cell self-renewal, promoted ES to TS transition and maintenance of TS cell stem-ness, but inhibited TS cell differentiation into both syncytial and EVT pathways.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Diferenciación Celular , Decorina/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Trofoblastos/metabolismo , Línea Celular Transformada , Femenino , Células Madre Embrionarias Humanas/citología , Humanos , Embarazo , Primer Trimestre del Embarazo , Trofoblastos/citologíaRESUMEN
G-protein-coupled receptors (GPCRs, also called seven-transmembrane or heptahelical receptors) are a superfamily of cell surface receptor proteins that bind to many extracellular ligands and transmit signals to an intracellular guanine nucleotide-binding protein (G-protein). When a ligand binds, the receptor activates the attached G-protein by causing the exchange of Guanosine-5'-triphosphate (GTP) for guanosine diphosphate (GDP). They play a major role in many physiological functions, as well as in the pathology of many diseases, including cancer progression and metastasis. Only a few GPCR members have been exploited as targets for developing drugs with therapeutic benefit in cancer. Present review briefly summarizes the signaling pathways utilized by the EP (prostaglandin E receptor) family of GPCR, their physiological and pathological roles in carcinogenesis, with special emphasis on the roles of EP4 in breast cancer progression. We make a case for EP4 as a promising newer therapeutic target for treating breast cancer. We show that an aberrant over-expression of cyclooxygenase (COX)-2, which is an inflammation-associated enzyme, occurring in 40-50% of breast cancer patients leads to tumor progression and metastasis due to multiple cellular events resulting from an increased prostaglandin (PG) E2 production in the tumor milieu. They include inactivation of host anti-tumor immune cells, such as Natural Killer (NK) and T cells, increased immuno-suppressor function of tumor-associated macrophages, promotion of tumor cell migration, invasiveness and tumor-associated angiogenesis, due to upregulation of multiple angiogenic factors including Vascular Endothelial Growth Factor (VEGF)-A, increased lymphangiogenesis (due to upregulation of VEGF-C/D), and a stimulation of stem-like cell (SLC) phenotype in cancer cells. All of these events were primarily mediated by activation of the Prostaglandin (PG) E receptor EP4 on tumor or host cells. We show that selective EP4 antagonists (EP4A) could mitigate all of these events tested with cells in vitro as well as in vivo in syngeneic COX-2 expressing mammary cancer bearing mice or immune-deficient mice bearing COX-2 over-expressing human breast cancer xenografts. We suggest that EP4A can avoid thrombo-embolic side effects of long term use of COX-2 inhibitors by sparing cardio-protective roles of PGI2 via IP receptor activation or PGE2 via EP3 receptor activation. Furthermore, we identified two COX-2/EP4 induced oncogenic and SLC-stimulating microRNAs-miR526b and miR655, one of which (miR655) appears to be a potential blood biomarker in breast cancer patients for monitoring SLC-ablative therapies, such as with EP4A. We suggest that EP4A will likely produce the highest benefit in aggressive breast cancers, such as COX-2 expressing triple-negative breast cancers, when combined with other newer agents, such as inhibitors of programmed cell death (PD)-1 or PD-L1.
Asunto(s)
Proteínas de Neoplasias , Subtipo EP4 de Receptores de Prostaglandina E , Neoplasias de la Mama Triple Negativas , Dinoprostona/genética , Dinoprostona/metabolismo , Femenino , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND: Lymphatic metastasis, facilitated by lymphangiogenesis is a common occurrence in breast cancer, the molecular mechanisms remaining incompletely understood. We had earlier shown that cyclooxygenase (COX)-2 expression by human or murine breast cancer cells promoted lymphangiogenesis and lymphatic metastasis by upregulating VEGF-C/D production by tumor cells or tumor-associated macrophages primarily due to activation of the prostaglandin receptor EP4 by endogenous PGE2. It is not clear whether tumor or host-derived PGE2 has any direct effect on lymphangiogenesis, and if so, whether EP4 receptors on lymphatic endothelial cells (LEC) play any role. METHODS: Here, we address these questions employing in vitro studies with a COX-2-expressing and VEGF-C/D-producing murine breast cancer cell line C3L5 and a rat mesenteric (RM) LEC line and in vivo studies in nude mice. RESULTS: RMLEC responded to PGE2, an EP4 agonist PGE1OH, or C3L5 cell-conditioned media (C3L5-CM) by increased proliferation, migration and accelerated tube formation on growth factor reduced Matrigel. Native tube formation by RMLEC on Matrigel was abrogated in the presence of a selective COX-2 inhibitor or an EP4 antagonist. Addition of PGE2 or EP4 agonist, or C3L5-CM individually in the presence of COX-2 inhibitor, or EP4 antagonist, restored tube formation, reinforcing the role of EP4 on RMLEC in tubulogenesis. These results were partially duplicated with a human dermal LEC (HMVEC-dLyAd) and a COX-2 expressing human breast cancer cell line MDA-MB-231. Knocking down EP4 with shRNA in RMLEC abrogated their tube forming capacity on Matrigel in the absence or presence of PGE2, EP4 agonist, or C3L5-CM. RMLEC tubulogenesis following EP4 activation by agonist treatment was dependent on PI3K/Akt and Erk signaling pathways and VEGFR-3 stimulation. Finally in a directed in vivo lymphangiogenesis assay (DIVLA) we demonstrated the lymphangiogenic as well as angiogenic capacity of PGE2 and EP4 agonist in vivo. DISCUSSION/CONCLUSIONS: These results demonstrate the roles of tumor as well as host-derived PGE2 in inducing lymphangiogenesis, at least in part, by activating EP4 and VEGFR-3 on LEC. EP4 being a common target on both tumor and host cells contributing to tumor-associated lymphangiogenesis reaffirms the therapeutic value of EP4 antagonists in the intervention of lymphatic metastasis in breast cancer.
Asunto(s)
Dinoprostona/metabolismo , Células Endoteliales/metabolismo , Linfangiogénesis/fisiología , Neoplasias Mamarias Experimentales/patología , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Células Endoteliales/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Desnudos , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Benzo(a)pyrene [B(a)P] is an environmental toxicant that alters the steroidogenic profile of testis and induces testicular dysfunction. In the present study, we have investigated the molecular signaling of B(a)P and the ameliorative potential of the natural aryl hydrocarbon receptor (AhR) antagonist and antioxidant, resveratrol, on B(a)P-induced male reproductive toxicity. Studies showed that B(a)P treatment resulted in p38 MAPK activation and increased inducible nitric oxide synthase (iNOS) production along with testicular apoptosis and steroidogenic dysfunction. Resveratrol cotreatment maintained testicular redox potential, increased serum testosterone level and enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3ßHSD, 17ßHSD) and prevented subsequent onset of apoptosis. Resveratrol cotreatment resulted inhibition of testicular cytochrome P4501A1 (CYP1A1) expression, which is the major B(a)P metabolizing agent for BPDE-DNA adduct formation. Resveratrol also significantly decreased the B(a)P-induced AhR protein level, its nuclear translocation and subsequent promoter activation, thereby decreased the expression of CYP1A1. Resveratrol also down-regulated B(a)P-induced testicular iNOS production through suppressing the activation of p38 MAPK and ATF2, thus improved the oxidative status of the testis and prevented apoptosis. Our findings cumulatively suggest that resveratrol inhibits conversion of B(a)P into BPDE by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.
Asunto(s)
Antioxidantes/uso terapéutico , Benzo(a)pireno/antagonistas & inhibidores , Suplementos Dietéticos , Contaminantes Ambientales/antagonistas & inhibidores , Infertilidad Masculina/prevención & control , Estilbenos/uso terapéutico , Testículo/efectos de los fármacos , Factor de Transcripción Activador 2/agonistas , Factor de Transcripción Activador 2/antagonistas & inhibidores , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antioxidantes/efectos adversos , Apoptosis/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Benzo(a)pireno/administración & dosificación , Benzo(a)pireno/toxicidad , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Suplementos Dietéticos/efectos adversos , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/administración & dosificación , Contaminantes Ambientales/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Infertilidad Masculina/inducido químicamente , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas Wistar , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Resveratrol , Estilbenos/efectos adversos , Testículo/metabolismo , Testículo/patología , Testosterona/agonistas , Testosterona/antagonistas & inhibidores , Testosterona/sangreRESUMEN
BACKGROUND: Decorin, a leucine-rich proteoglycan that is produced by decidual cells, limits invasion and endovascular differentiation of extravillous trophoblast cells during early placentation by binding to multiple tyrosine kinase receptors, in particular, vascular endothelial growth factor receptor-2. OBJECTIVE: Because many studies have reported an association between poor trophoblast invasion and endovascular differentiation with preeclampsia, the studies reported here tested (1) whether decorin over-expression in the chorionic villi and/or basal decidua is associated with preeclampsia and, if so, (2) whether this association results in a hypoinvasive placenta, and (3) whether elevated plasma decorin concentration in the second trimester is a predictive biomarker for preeclampsia. STUDY DESIGN: Decorin messenger RNA expression was measured with quantitative polymerase chain reaction at the tissue level and with in situ hybridization at the cellular level using (35)S-labeled antisense complimentary RNA probe in placentas from healthy control subjects and subjects with preeclampsia (14 each, 23-40 weeks of gestation). Tissue sections of the same placentas were also immunostained for decorin protein. A decorin over-expressing human endometrial stromal cell line was tested for invasion-regulatory effects on an invasive first-trimester extravillous trophoblast cell line HTR-8/SVneo plated in cocultures that were separated by a semipermeable membrane. Furthermore, we conducted retrospective measurements of plasma decorin levels during the second trimester (15-18 weeks of gestation) in a cohort of 28 body mass index-matched pairs of control subjects and subjects with preeclampsia before the onset of clinical disease. RESULTS: First, decorin messenger RNA expression at the cellular level measured with in situ hybridization exhibited profoundly higher expression levels in basal plate decidual cells within the placentas from preeclamptic subjects than those from control subjects at all gestational ages, whereas no difference between the 2 subject groups was noted in villus mesenchymal cells. Similarly decorin messenger RNA expression at the tissue level in chorionic villi (primarily resulting from fetally derived mesenchymal cells) did not differ significantly between control and preeclampsia placentas. These findings were validated with immunostaining for decorin protein. Second, knocking down decorin gene in a decorin over-expressing endometrial cell line (used as an in vitro surrogate of decorin over-expressing decidual cells) in cocultures with extravillous trophoblast cells abrogated its invasion-restraining actions on trophoblast cells, which indicated paracrine contribution of decorin over-expressing decidua to the poor trophoblast invasiveness in situ. Finally, retrospective measurement of plasma decorin levels during the second trimester in 28 body mass index-matched pairs of control subjects and subjects with preeclampsia revealed elevated plasma decorin levels in all subjects with preeclampsia in all body mass index groups. A receiver operating characteristic curve analysis revealed strong diagnostic performance of plasma decorin in the prediction of preeclampsia status. Although there was no significant gestational age-related change in decorin levels during the second trimester in control or subjects with preeclampsia, we found that plasma decorin had a significant inverse relationship with body mass index or bodyweight. CONCLUSION: We conclude that decorin over-expression by basal decidual cells is associated with hypoinvasive phenotype and poor endovascular differentiation of trophoblast cells in preeclampsia and that elevated plasma decorin concentration is a potential predictive biomarker for preeclampsia before the onset of clinical signs.
Asunto(s)
Decidua/metabolismo , Decorina/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Decidua/citología , Decorina/genética , Femenino , Humanos , Hibridación in Situ , Reacción en Cadena de la Polimerasa , Embarazo , Segundo Trimestre del Embarazo , ARN Mensajero/metabolismoRESUMEN
The objective of the present review is to synthesize the information on the cellular and molecular players responsible for maintaining a homeostatic balance between a naturally invasive human placenta and the maternal uterus in pregnancy; to review the roles of decorin (DCN) as a molecular player in this homeostasis; to list the common maladies associated with a break-down in this homeostasis, resulting from a hypo-invasive or hyper-invasive placenta, and their underlying mechanisms. We show that both the fetal components of the placenta, represented primarily by the extravillous trophoblast, and the maternal component represented primarily by the decidual tissue and the endometrial arterioles, participate actively in this balance. We discuss the process of uterine angiogenesis in the context of uterine arterial changes during normal pregnancy and preeclampsia. We compare and contrast trophoblast growth and invasion with the processes involved in tumorigenesis with special emphasis on the roles of DCN and raise important questions that remain to be addressed. Decorin (DCN) is a small leucine-rich proteoglycan produced by stromal cells, including dermal fibroblasts, chondrocytes, chorionic villus mesenchymal cells and decidual cells of the pregnant endometrium. It contains a 40 kDa protein core having 10 leucine-rich repeats covalently linked with a glycosaminoglycan chain. Biological functions of DCN include: collagen assembly, myogenesis, tissue repair and regulation of cell adhesion and migration by binding to ECM molecules or antagonising multiple tyrosine kinase receptors (TKR) including EGFR, IGF-IR, HGFR and VEGFR-2. DCN restrains angiogenesis by binding to thrombospondin-1, TGFß, VEGFR-2 and possibly IGF-IR. DCN can halt tumor growth by antagonising oncogenic TKRs and restraining angiogenesis. DCN actions at the fetal-maternal interface include restraint of trophoblast migration, invasion and uterine angiogenesis. We demonstrate that DCN overexpression in the decidua is associated with preeclampsia (PE); this may have a causal role in PE by compromising endovascular differentiation of the trophoblast and uterine angiogenesis, resulting in poor arterial remodeling. Elevated DCN level in the maternal blood is suggested as a potential biomarker in PE.
Asunto(s)
Movimiento Celular , Decorina/metabolismo , Endometrio/irrigación sanguínea , Neovascularización Fisiológica , Preeclampsia/metabolismo , Preeclampsia/patología , Trofoblastos/patología , Animales , Endometrio/patología , Femenino , Humanos , EmbarazoRESUMEN
Decorin (DCN) is a leucine-rich, TGF-ß binding proteoglycan produced by mesenchymal cells including chondrocytes, dermal fibroblasts, and uterine decidual cells. It exerts multiple physiological functions including collagen fibrillogenesis, myogenesis, angiostasis, and restraining placental invasiveness. We discovered that decidua-derived DCN restrains proliferation, migration, and invasion of extravillous trophoblast (EVT) cells of the human placenta in a TGF-ß-independent manner. These functions were differentially mediated by binding of DCN to multiple tyrosine kinase receptors (TKR) including EGFR, IGFR1, and VEGFR2. DCN blocked VEGFR-2 dependent EVT cell migration and endovascular differentiation by inhibiting P38MAPK and ERK1/2 pathways.We identified the avid VEGFR2 binding site in DCN protein as a 12 amino acids (LGTNPLKSSGIE) span in the Leucine-rich-repeat (LRR) 5 region of domain III. A single amino acid mutation (substitution of K to A) of DCN at this site abrogated VEGFR-2- dependent DCN actions. Also, DCN mRNA expression, measured with in situ hybridization, was selectively upregulated in decidual cells in placentas from mothers suffering from pre-eclampsia (PE), whereas the expression levels remained unchanged in chorionic villus mesenchymal cells. This difference between PE and control placentas was present at all gestational ages, indicating the pathogenic role of DCN in PE. We hypothesize that increased blood DCN levels could be a candidate biomarker for PE.
Asunto(s)
Decidua/inmunología , Decorina/inmunología , Implantación del Embrión/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Preeclampsia/inmunología , Trofoblastos/inmunología , Animales , Biomarcadores/sangre , Decidua/metabolismo , Decidua/patología , Decorina/sangre , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Preeclampsia/sangre , Embarazo , Proteínas Tirosina Quinasas Receptoras/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patologíaRESUMEN
Multiple mechanisms have been proposed by which tumors induce T cell apoptosis to circumvent tumor immune-surveillance. Although sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) have long been known to regulate intracellular Ca(2+) homeostasis, few studies have examined the role of SERCA in processes of T lymphocyte survival and activation. In this context it remains largely unexplored as to how tumors jeopardize SERCA function to disable T cell-mediated anti-tumor immunity. Here, we show that human CD4(+) T cells in the presence of tumor conditions manifested an up-regulation of SERCA3 expression that resulted in development of endoplasmic reticulum stress leading to CD4(+) T cell apoptosis. Prostaglandin E(2) produced by the tumor cell plays a critical role in up-regulating SERCA3 by enhancing the binding of its transcription factor Sp1. Gene manipulation and pharmacological approaches further established that an increase in SERCA expression also resulted in subsequent inhibition of PKCα and -θ and retention of NFκB in the cytosol; however, down-modulation of SERCA3 expression by a dihydropyrimidone derivative, ethyl-4-(3-nitro)-phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate (nifetepimine), protected the CD4(+) T cells from tumor-induced apoptosis. In fact, nifetepimine-mediated restoration of PKC activity resulted in nuclear translocation of p65NFκB, thereby ensuring its survival. Studies further undertaken in a tumor-bearing mice model revalidated the immunoprotective role of nifetepimine. Our present study thus strongly suggests that imbalance in cellular calcium homeostasis is an important factor leading to CD4(+) T cell death during cancer and holds promise that nifetepimine may have the potential to be used as an immunorestoring agent in cancer bearers.
Asunto(s)
Neoplasias de la Mama/enzimología , Linfocitos T CD4-Positivos/metabolismo , Calcio/metabolismo , Factores Inmunológicos/farmacología , Proteínas de Neoplasias/metabolismo , Pirimidinonas/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Microambiente Tumoral/efectos de los fármacos , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Linfocitos T CD4-Positivos/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Dinoprostona/genética , Dinoprostona/inmunología , Dinoprostona/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/inmunología , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/inmunología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Trasplante de Neoplasias , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/inmunología , Proteína Quinasa C-alfa/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/inmunología , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/inmunología , Factor de Transcripción Sp1/metabolismo , Trasplante Heterólogo , Microambiente Tumoral/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
A regioselective N1-alkylation of 3,4-dihydropyrimidin-2(1H)-ones using a very efficient mild base Cs(2)CO(3) and alkyl halides at room temperature has been reported. The selectivity of this methodology is excellent and the yields of the alkylated products are very good. Furthermore inhibitory action of both the 3,4-dihydropyrimidin-2(1H)-ones and the N1-alkylated derivatives were tested on Ca(2+)-ATPase, which revealed that the parent compounds can act as Ca(2+)-ATPase inhibitors whereas the N1-alkylated derivatives are inefficient for this purpose.
Asunto(s)
ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Pirimidinonas/síntesis química , Pirimidinonas/farmacología , Alquilación , Animales , ATPasas Transportadoras de Calcio/química , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Modelos Moleculares , Conformación Molecular , Pirimidinonas/química , Estereoisomerismo , Especificidad por SustratoRESUMEN
Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.
Asunto(s)
Reacción Acrosómica , Cabras/fisiología , Proteínas de Plasma Seminal/fisiología , Capacitación Espermática , Espermatozoides/metabolismo , Espermatozoides/fisiología , Reacción Acrosómica/fisiología , Animales , Epidídimo/metabolismo , Células Epiteliales/metabolismo , Citometría de Flujo , Cabras/metabolismo , Masculino , Microscopía Fluorescente , Análisis de Semen , Proteínas de Plasma Seminal/metabolismo , Capacitación Espermática/fisiología , Espermatogénesis/fisiología , Distribución TisularRESUMEN
Synthesis of a series of novel (6-deoxy-glycopyranosid-6-yl) sulfone derivatives has been achieved using a general synthetic strategy. Yields were excellent in every case. The synthetic compounds were evaluated for their biological potential against Ca2+-ATPase, an important enzyme involves in transporting Ca2+ across the cell membranes.
Asunto(s)
ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glucósidos/síntesis química , Glucósidos/farmacología , Sulfonas/síntesis química , Sulfonas/farmacología , Animales , Conformación de Carbohidratos , Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Glucósidos/química , Cabras , Masculino , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimología , Relación Estructura-Actividad , Sulfonas/químicaRESUMEN
Protein tyrosine phosphorylation is a key event accompanying sperm capacitation. Although this signaling cascade generates an array of tyrosine-phosphorylated polypeptides, their molecular characterization is still limited. It is necessary to differentiate the localization of the tyrosine-phosphorylated proteins in spermatozoa to understand the link between the different phosphorylated proteins and the corresponding regulated sperm function. cAMP plays a pivotal role in the regulation of tyrosine phosphorylation. The intracellular cAMP levels were raised in goat spermatozoa by the addition of the phosphodiesterase inhibitor, IBMX in conjugation with caffeine. Tyrosine phosphorylation was significantly up-regulated following treatment with these two reagents. Treatment of caudal spermatozoa with IBMX and caffeine, time dependent up-regulated phosphorylation of the protein of molecular weights 50 and 200 kDa was observed. Increased phosphorylation was observed with a combination of IBMX and caffeine treatment. Tyrosine phosphorylation in caput spermatozoa was not affected significantly under these conditions. The expression level of tyrosine kinase in sperm was examined with specific inhibitors and with anti-phosphotyrosine antibody. The indirect immunofluorescence staining was carried out on ethanol permeabilized sperm using anti-phosphotyrosine antibody. Western blot analysis was done using two separate PKA antibodies: anti-PKA catalytic and anti-PKA RIalpha. Almost no difference was found in the intracellular presence of the PKA RIalpha and RIIalpha subunits in caput and caudal epididymal spermatozoa. However, the catalytic subunit seemed to be present in higher amount in caudal spermatozoa. The results show that caprine sperm displays an enhancement of phosphorylation in the tyrosine residues of specific proteins under in vitro capacitation conditions.