RESUMEN

After menstruation the uterine spiral arteries are repaired through angiogenesis. This process is tightly regulated by the paracrine communication between endometrial stromal cells (EnSCs) and endothelial cells. Any molecular aberration in these processes can lead to complications in pregnancy including miscarriage or preeclampsia (PE). Placental growth factor (PlGF) is a known contributing factor for pathological angiogenesis but the mechanisms remain poorly understood. In this study, we investigated whether PlGF contributes to pathological uterine angiogenesis by disrupting EnSCs and endothelial paracrine communication. We observed that PlGF mediates a tonicity-independent activation of nuclear factor of activated T cells 5 (NFAT5) in EnSCs. NFAT5 activated downstream targets including SGK1, HIF-1α and VEGF-A. In depth characterization of PlGF - conditioned medium (CM) from EnSCs using mass spectrometry and ELISA methods revealed low VEGF-A and an abundance of extracellular matrix organization associated proteins. Secreted factors in PlGF-CM impeded normal angiogenic cues in endothelial cells (HUVECs) by downregulating Notch-VEGF signaling. Interestingly, PlGF-CM failed to support human placental (BeWo) cell invasion through HUVEC monolayer. Inhibition of SGK1 in EnSCs improved angiogenic effects in HUVECs and promoted BeWo invasion, revealing SGK1 as a key intermediate player modulating PlGF mediated anti-angiogenic signaling. Taken together, perturbed PlGF-NFAT5-SGK1 signaling in the endometrium can contribute to pathological uterine angiogenesis by negatively regulating EnSCs-endothelial crosstalk resulting in poor quality vessels in the uterine microenvironment. Taken together the signaling may impact on normal trophoblast invasion and thus placentation and, may be associated with an increased risk of complications such as PE.

Asunto(s)

Endometrio , Neovascularización Patológica , Factor de Crecimiento Placentario , Preeclampsia , Proteínas Serina-Treonina Quinasas , Factores de Transcripción , Femenino , Humanos , Embarazo , Endometrio/metabolismo , Endometrio/irrigación sanguínea , Ensayo de Inmunoadsorción Enzimática , Proteínas Inmediatas-Precoces/metabolismo , Neovascularización Patológica/metabolismo , Factor de Crecimiento Placentario/metabolismo , Preeclampsia/metabolismo , Preeclampsia/fisiopatología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Células del Estroma/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

Cardiac fibroblasts (CF) are mesenchymal-type cells responsible for maintaining the homeostasis of the heart's extracellular matrix (ECM). Their dysfunction leads to excessive secretion of ECM proteins, tissue stiffening, impaired nutrient and oxygen exchange, and electrical abnormalities in the heart. Additionally, CF act as sentinel cells in the cardiac tissue microenvironment, responding to various stimuli that may affect heart function. Deleterious stimuli induce an inflammatory response in CF, increasing the secretion of cytokines such as IL-1ß and TNF-α and the expression of cell adhesion molecules like ICAM1 and VCAM1, initially promoting damage resolution by recruiting immune cells. However, constant harmful stimuli lead to a chronic inflammatory process and heart dysfunction. Therefore, it is necessary to study the mechanisms that govern CF inflammation. NFκB is a key regulator of the cardiac inflammatory process, making the search for mechanisms of NFκB regulation and CF inflammatory response crucial for developing new treatment options for cardiovascular diseases. SGK1, a serine-threonine protein kinase, is one of the regulators of NFκB and is involved in the fibrotic effects of angiotensin II and aldosterone, as well as in CF differentiation. However, its role in the CF inflammatory response is unknown. On the other hand, many bioactive natural products have demonstrated anti-inflammatory effects, but their role in CF inflammation is unknown. One such molecule is boldine, an alkaloid obtained from Boldo (Peumus boldus), a Chilean endemic tree with proven cytoprotective effects. However, its involvement in the regulation of SGK1 and CF inflammation is unknown. In this study, we evaluated the role of SGK1 and boldine in the inflammatory response in CF isolated from neonatal Sprague-Dawley rats. The involvement of SGK1 was analyzed using GSK650394, a specific SGK1 inhibitor. Our results demonstrate that SGK1 is crucial for LPS- and IFN-γ-induced inflammatory responses in CF (cytokine expression, cell adhesion molecule expression, and leukocyte adhesion). Furthermore, a conditioned medium (intracellular content of CF subject to freeze/thaw cycles) was used to simulate a sterile inflammation condition. The conditioned medium induced a potent inflammatory response in CF, which was completely prevented by the SGK1 inhibitor. Finally, our results indicate that boldine inhibits both SGK1 activation and the CF inflammatory response induced by LPS, IFN-γ, and CF-conditioned medium. Taken together, our results position SGK1 as an important regulator of the CF inflammatory response and boldine as a promising anti-inflammatory drug in the context of cardiovascular diseases.

Asunto(s)

Aporfinas , Fibroblastos , Proteínas Inmediatas-Precoces , FN-kappa B , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Animales , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ratas , Aporfinas/farmacología , Inflamación/metabolismo , Inflamación/patología , Miocardio/patología , Miocardio/metabolismo , Células Cultivadas , Ratas Sprague-Dawley

RESUMEN

Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for sodium homeostasis and implicated in arterial hypertension (HTN). Changes in the lipid composition of the plasma membrane can significantly impact cellular processes related to physiological functions. We hypothesized that the observed overexpression of ENaC in neutrophils from HTN patients might result from alterations in the structuring domains within the plasma membrane, disrupting the endocytic processes responsible for ENaC retrieval. This study assessed the structural lipid composition of neutrophil plasma membranes from HTN patients along with the expression patterns of key elements regulating ENaC at the plasma membrane. Our findings suggest alterations in microdomain structure and SGK1 kinase activity, which could prolong ENaC presence on the plasma membrane. Additionally, we propose that the proteasomal and lysosomal degradation pathways are insufficient to diminish ENaC presence at the plasma membrane in HTN. These results highlight the importance of understanding ENaC retrieval mechanisms and suggest that targeting these mechanisms could provide insights for developing drugs to prevent and treat HTN.

Asunto(s)

Membrana Celular , Endocitosis , Canales Epiteliales de Sodio , Hipertensión , Neutrófilos , Canales Epiteliales de Sodio/metabolismo , Humanos , Neutrófilos/metabolismo , Hipertensión/metabolismo , Hipertensión/patología , Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Masculino , Femenino , Proteínas Inmediatas-Precoces/metabolismo , Persona de Mediana Edad , Microdominios de Membrana/metabolismo

RESUMEN

Prostate cancer (PCa) has a high prevalence and represents an important health problem, with an increased risk of metastasis. With the advance of CRISPR-Cas9 genome editing, new possibilities have been created for investigating PCa. The technique is effective in knockout oncogenes, reducing tumor resistance. MMP9 and miR-21 target genes are associated with PCa progression; therefore, we evaluated the MMP-9 and miR-21 targets in PCa using the CRISPR-Cas9 system. Single guide RNAs (sgRNAs) of MMP9 and miR-21 sequences were inserted into a PX-330 plasmid, and transfected in DU145 and PC-3 PCa cell lines. MMP9 and RECK expression was assessed by qPCR, WB, and IF. The miR-21 targets, integrins, BAX and mTOR, were evaluated by qPCR. Flow cytometry was performed with Annexin5, 7-AAD and Ki67 markers. Invasion assays were performed with Matrigel. The miR-21 CRISPR-Cas9-edited cells upregulated RECK, MARCKS, BTG2, and PDCD4. CDH1, ITGB3 and ITGB1 were increased in MMP9 and miR-21 CRISPR-Cas9-edited cells. Increased BAX and decreased mTOR were observed in MMP9 and miR-21 CRISPR-Cas9-edited cells. Reduced cell proliferation, increased apoptosis and low invasion in MMP9 and miR-21 edited cells was observed, compared to Scramble. CRISPR-Cas9-edited cells of miR-21 and MMP9 attenuate cell proliferation, invasion and stimulate apoptosis, impeding PCa evolution.

Asunto(s)

Proteínas Inmediatas-Precoces , MicroARNs , Neoplasias de la Próstata , Masculino , Humanos , Edición Génica , Sistemas CRISPR-Cas/genética , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , ARN Guía de Sistemas CRISPR-Cas , Proteína X Asociada a bcl-2/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , MicroARNs/genética , MicroARNs/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Supresoras de Tumor/genética , Proteínas de Unión al ARN/metabolismo

RESUMEN

The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl- ) accumulation. The anion Cl- , acting as a second messenger, stimulates the secretion of interleukin-1ß (IL-1ß), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl- concentration, showing maximal expression and activity at 75 mM Cl- , in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125 mM Cl- . The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl- -stimulated IL-1ß mRNA expression and partially the IL-1ß secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75 mM Cl- , a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1ß mRNA response to Cl- and the IL-1ß secretion, interrupting the autocrine IL-1ß loop. The results suggest that Cl- effects are mediated by SGK1, in which under Cl- modulation stimulates the secretion of mature IL-1ß, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1ß itself, through autocrine signalling.

Asunto(s)

Caspasa 1/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Interleucina-1beta/metabolismo , Espacio Intracelular/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Inhibidores de Caspasas/farmacología , Línea Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Dipéptidos/farmacología , Retroalimentación Fisiológica , Furanos/farmacología , Humanos , Proteínas Inmediatas-Precoces/genética , Indenos/farmacología , Interleucina-1beta/genética , Mutación/genética , Nigericina/farmacología , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Sulfonamidas/farmacología , para-Aminobenzoatos/farmacología

RESUMEN

In recent years, the role of anti-proliferative TOB proteins in the regulation of immune response by inhibiting T cell activation has been demonstrated. Nevertheless, no previous studies have explored their expression in patients with IBD. The aim of the study was to characterize the gene and protein expression of the TOB/BTG family in intestinal tissue of patients with IBD. This is an observational and cross-sectional study that included 63 IBD patients. Gene expression of TOB/BTG family was measured by RT-PCR. Protein expression of TOB/CD16 and BTG/Ki-67 was evaluated by immunohistochemistry. TOB/BTG family mRNAs were detected and quantitated by RT-qPCR in rectal and ileum biopsies from UC patients and CD patients, respectively, and non-inflammatory control tissues. Results showed that TOB1 and BTG1 gene expression was decreased in the colonic mucosa from patients with UC compared with the control group. The TOB2 and BTG2 genes were over-expressed in the colonic mucosa of patients with UC in remission compared with the active UC and control group. The high TOB2 gene expression was associated with histological remission (P = .01). TOB1/CD16, TOB2/CD16, BTG1/Ki-67, BTG2/Ki-67 and BTG4/Ki-67 single and double positive cells were mostly NK, macrophages, epithelial cells, connective tissue cells and perivascular inflammatory infiltrates in tissues from patients with UC and CD. This is the first depiction of the TOB/BTG family gene and protein expression in rectal and ileum tissues by a CD16+ subpopulation in IBD.

Asunto(s)

Proteínas Inmediatas-Precoces/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adulto , Proliferación Celular/fisiología , Colitis/metabolismo , Colon/metabolismo , Estudios Transversales , Células Epiteliales/metabolismo , Femenino , Expresión Génica/fisiología , Humanos , Mucosa Intestinal/metabolismo , Antígeno Ki-67/metabolismo , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Receptores de IgG/metabolismo

RESUMEN

Herpesviruses are predicted to express more than 80 proteins during their infection cycle. The proteins synthesized by the immediate early genes and early genes target signaling pathways in host cells that are essential for the successful initiation of a productive infection and for latency. In this study, proteomic and phosphoproteomic tools showed the occurrence of changes in Madin-Darby bovine kidney cells at the early stage of the infection by bovine herpesvirus 1 (BoHV-1). Proteins that had already been described in the early stage of infection for other herpesviruses but not for BoHV-1 were found. For example, stathmin phosphorylation at the initial stage of infection is described for the first time. In addition, two proteins that had not been described yet in the early stages of herpesvirus infections in general were ribonuclease/angiogenin inhibitor and Rab GDP dissociation inhibitor beta. The biological processes involved in these cellular responses were repair and replication of DNA, splicing, microtubule dynamics, and inflammatory responses. These results reveal pathways that might be used as targets for designing antiviral molecules against BoHV-1 infection.

Asunto(s)

Infecciones por Herpesviridae/metabolismo , Herpesvirus Bovino 1/patogenicidad , Proteómica/métodos , Proteínas Virales/metabolismo , Animales , Bovinos , Línea Celular , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Espectrometría de Masas , Fosforilación , Mapas de Interacción de Proteínas , Estatmina/metabolismo , Replicación Viral

RESUMEN

Abstract Introduction: Serum- and glucocorticoid-inducible kinase 3, a serine/threonine kinase that functions downstream of the PI3K signaling pathway, plays a critical role in neoplastic processes. It is expressed by various tumors and contributes to carcinogenesis. Objective: The objective was to investigate serum- and glucocorticoid-inducible kinase 3 expression in nasopharyngeal carcinoma, to study the anti-tumor effects of serum- and glucocorticoid-inducible kinase 3 shRNA by inhibiting its expression in nasopharyngeal carcinoma cells and to discuss the potential implications of our findings. Methods: Serum- and glucocorticoid-inducible kinase 3 protein expression in nasopharyngeal carcinoma cell lines (CNE-1, CNE-2, HNE-1, HONE-1, and SUNE-1) and the human immortalized nasopharyngeal epithelium cell line NP69 were assayed by western blotting. Serum- and glucocorticoid-inducible kinase 3 expression in 42 paraffin-embedded nasopharyngeal carcinoma tissues were performed by immunohistochemistry. MTT assay, flow cytometry, and scratch tests were performed after CNE-2 cells were transfected with the best serum- and glucocorticoid-inducible kinase 3 shRNA plasmid selected by western blotting using lipofectamine to study its effect on cell proliferation, apoptosis, and migration. Results: Serum- and glucocorticoid-inducible kinase 3 was overexpressed in human nasopharyngeal carcinoma tissues and cells. Serum- and glucocorticoid-inducible kinase 3 expression decreased markedly after CNE-2 cells were transfected with the serum- and glucocorticoid-inducible kinase 3 shRNA, leading to strong inhibition of cell proliferation and migration. In addition, the apoptosis rate increased in CNE-2 cells after serum- and glucocorticoid-inducible kinase 3 knockdown. Conclusion: Serum- and glucocorticoid-inducible kinase 3 expression was more frequently observed as the nasopharyngeal epithelium progresses from normal tissue to carcinoma. This suggests that serum- and glucocorticoid-inducible kinase 3 contributes to the multistep process of NPC carcinogenesis. Serum- and glucocorticoid-inducible kinase 3 represents a target for nasopharyngeal carcinoma therapy, and a basis exists for the further investigation of this adjuvant treatment modality for nasopharyngeal carcinoma.

---

Resumo Introdução: A quinase 3 sérica induzida por glicocorticoide, uma serina/treonina quinase que funciona downstream da via de sinalização PI3K, desempenha um papel crítico nos processos neoplásicos. É expressa por vários tumores e contribui para a carcinogênese. Objetivo: Investigar a expressão de quinase 3 sérica induzida por glicocorticoide no carcinoma nasofaríngeo, estudar os efeitos antitumorais do shRNA da quinase 3 sérica induzida por glicocorticoide, que inibem sua expressão em células de carcinoma nasofaríngeo, e discutir as implicações potenciais de nossos achados. Método: A expressão de proteína quinase 3 sérica induzida por glicocorticoide em linhagens de células de carcinoma nasofaríngeo (CNE-1, CNE-2, HNE-1, HONE-1 e SUNE-1) e a linhagem de células humanas imortalizadas do epitélio nasofaríngeo NP69 foram avaliadas por Western blot. A expressão da quinase 3 sérica induzida por glicocorticoide em 42 tecidos de CNF embebidos em parafina foi feita por imuno-histoquímica. Testes com MTT, citometria de fluxo e testes de raspagem foram feitos após as células CNE-2 terem sido transfectadas com o melhor plasmídeo shRNA da quinase 3 sérica induzida por glicocorticoide selecionado por Western blot, com o uso de lipofectamina para estudar seu efeito na proliferação, apoptose e migração celular. Resultados: Foi observada uma sobre-expressão da quinase 3 sérica induzida por glicocorticoide em tecidos e células de carcinoma nasofaríngeo humanas. A expressão de quinase 3 sérica induzida por glicocorticoide diminuiu acentuadamente após as células CNE-2 terem sido transfectadas com o shRNA da quinase 3 sérica induzida por glicocorticoide, conduzindo a forte inibição de proliferação e migração celular. Além disso, a taxa de apoptose aumentou nas células CNE-2 após o knockdown da quinase 3 sérica induzida por glicocorticoide. Conclusão: A expressão de quinase 3 sérica induzida por glicocorticoide foi observada com maior frequência à medida que o epitélio nasofaríngeo progride de tecido normal para carcinoma. Isso sugere que a quinase 3 sérica induzida por glicocorticoide contribui para o processo multietapas da carcinogênese do carcinoma nasofaríngeo. A quinase 3 sérica induzida por glicocorticoide representa um alvo para a terapia do carcinoma nasofaríngeo e há uma base para a investigação adicional dessa modalidade de tratamento adjuvante para o carcinoma nasofaríngeo.

Asunto(s)

Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Neoplasias Nasofaríngeas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Inmunohistoquímica , Movimiento Celular/efectos de los fármacos , Neoplasias Nasofaríngeas/patología , Nasofaringitis/metabolismo , Nasofaringitis/patología , Proteínas Serina-Treonina Quinasas/farmacología , Apoptosis , Proteínas Inmediatas-Precoces/farmacología , ARN Interferente Pequeño/metabolismo , Proliferación Celular/efectos de los fármacos , Carcinoma Nasofaríngeo/patología

RESUMEN

Chronic vasopressin secretion induced by recurrent mild heat stress exposure is significantly enhanced by limited rehydration with a fructose-containing beverage both in rodents and in humans. Moreover, this effect has been associated with upregulation of the polyol-fructokinase pathway and increased renal oxidative stress. Previously, we have shown that pharmacological inhibition of both V1a and V2 vasopressin receptors with conivaptan improved such renal alterations. The aim of this study was to evaluate the independent contributions of V1a and V2 receptors to the renal damage caused by mild heat stress and limited rehydration with a fructose-containing beverage. Osmotic minipumps were used to deliver either relcovaptan (0.64 mg/day) or tolvaptan (0.25 mg/day) in male Wistar rats for two weeks. Corresponding dilution vehicles were used as controls. To induce dehydration, rats were exposed to mild heat stress (37 °C for 1 h, Monday to Friday). All groups received a 10% fructose solution as a rehydration fluid for 2 h after mild heat stress. For the remainder of the day and on weekends, rats received tap water. The independent blockade of either the V1a or the V2 receptor prevented renal damage, reduced oxidative stress, and decreased plasma cortisol and systemic inflammation. However, the beneficial effects were regulated by different mechanisms. Tolvaptan inhibited polyol-fructokinase pathway overactivation, while relcovaptan prevented upregulation of the renin-angiotensin system and SGK1 expression. These data suggest that both V1a and V2 receptors participate in renal damage caused by heat stress-induced dehydration when fructose-containing beverages are used as rehydration fluids.

Asunto(s)

Bebidas/análisis , Fructosa/metabolismo , Respuesta al Choque Térmico , Receptores de Vasopresinas/metabolismo , Animales , Fluidoterapia , Respuesta al Choque Térmico/efectos de los fármacos , Hidrocortisona/sangre , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Indoles/farmacología , Corteza Renal/metabolismo , Masculino , Estrés Oxidativo/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pirrolidinas/farmacología , Ratas , Ratas Wistar , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Sistema Renina-Angiotensina/efectos de los fármacos , Temperatura , Tolvaptán/farmacología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

BACKGROUND: Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma with a poor prognosis owing to its resistance to radiation and chemotherapy. Thus, novel therapeutic strategies for SS are urgently required. Anlotinib, a new oral tyrosine kinase inhibitor, is designed to primarily inhibit multi-targets in vasculogenesis and angiogenesis. This study was designed to characterize its antitumor efficacy and possible mechanism in patients with advanced refractory synovial sarcoma. METHODS: Anlotinib's antitumor effect was evaluated in vivo and vitro. Downstream targets of anlotinib in treating synovial sarcoma were analyzed through microarray assay. Cell proliferation and apoptosis analyses were performed to evaluate the impact of candidate downstream gene depletion in synovial sarcoma cells. Microarray assay were carried out to investigate potential signal network related with candidate downstream gene. RESULTS: Anlotinib significantly suppresses synovial sarcoma proliferation in PDTX model and cell lines. Additionally, GINS1 (also named as PSF1, Partner of SLD Five 1), rather than other conventional gene target, was demonstrated to be a vital target of anlotinib's antitumor effect in synovial sarcoma through microarray assay. Expression of GINS1 was remarkably higher in synovial sarcoma tumor samples and related with poor outcome. Knockdown of GINS1 expression could remarkably inhibit proliferation and promote apoptosis in vitro. Meanwhile, through microarray assay, CITED2, EGR1, SGK1 and SPP1 were identified and further validated by qPCR/WB as downstream targets of GINS1. CONCLUSION: Anlotinib might suppress proliferation of SS through a novel downstream GINS1-regulated network which plays a vital function in SS proliferation and also demonstrated that targeting the GINS1-regulated signal pathway could be a potential strategy for management of SS.

Asunto(s)

Neoplasias Óseas/tratamiento farmacológico , Proteínas de Unión al ADN/efectos de los fármacos , Indoles/uso terapéutico , Proteínas de Neoplasias/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinolinas/uso terapéutico , Sarcoma Sinovial/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Neoplasias Óseas/genética , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Osteopontina/efectos de los fármacos , Osteopontina/genética , Osteopontina/metabolismo , Análisis por Matrices de Proteínas , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/análisis , Proteínas Represoras/efectos de los fármacos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Sarcoma Sinovial/genética , Transactivadores/efectos de los fármacos , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

Malignant gliomas are the most common types of incurable primary brain tumours. Therefore, to better clarify the aetiology and pathogenesis of the disease and analyse the risk factors involved, several researchers have highlighted a possible link to human cytomegalovirus (HCMV). Regarding this potential link, the numbers of studies and controversies concerning the relationship between HCMV infections and malignant gliomas have significantly increased. Therefore, we conducted a meta-analysis of observational studies to summarize and pool the available results on the association of HCMV in patients with glioma. Our meta-analysis was based on the PRISMA algorithm, using fixed/random models through STATA IC 13.1 software. Thus, 32 studies were included with a total of 2,190 participants/specimens (glioma, n = 1,871; non-glioma, n = 319). The overall estimate of combined HCMV frequency in patients with glioma was 63% (95% confidence interval [CI]: 56-70). There was an association between HCMV infection and glioma (adjusted OR = 3, 95% CI: 1.7-5.3). The pooled subgroup analysis of viral markers also showed a positive association between the pp65 protein (OR = 3.1, 95% CI: 1.8-5), and gB nucleic acids (OR = 3.1, 95% CI: 1.1-8). For the viral marker IE1-72 protein, the pooled frequency and association results were higher. However, there was no correlation of higher viral association according to the histological subtypes and low/high grade of gliomas. In conclusion, the available evidence suggests an association between HCMV and glioma. Consequently, precautions should be taken, as discussed in this report.

Asunto(s)

Neoplasias Encefálicas/virología , Infecciones por Citomegalovirus/patología , Citomegalovirus/aislamiento & purificación , Glioma/virología , Neoplasias Encefálicas/patología , Citomegalovirus/genética , Glioma/patología , Humanos , Proteínas Inmediatas-Precoces/genética

RESUMEN

INTRODUCTION: Serum- and glucocorticoid-inducible kinase 3, a serine/threonine kinase that functions downstream of the PI3K signaling pathway, plays a critical role in neoplastic processes. It is expressed by various tumors and contributes to carcinogenesis. OBJECTIVE: The objective was to investigate serum- and glucocorticoid-inducible kinase 3 expression in nasopharyngeal carcinoma, to study the anti-tumor effects of serum- and glucocorticoid-inducible kinase 3 shRNA by inhibiting its expression in nasopharyngeal carcinoma cells and to discuss the potential implications of our findings. METHODS: Serum- and glucocorticoid-inducible kinase 3 protein expression in nasopharyngeal carcinoma cell lines (CNE-1, CNE-2, HNE-1, HONE-1, and SUNE-1) and the human immortalized nasopharyngeal epithelium cell line NP69 were assayed by western blotting. Serum- and glucocorticoid-inducible kinase 3 expression in 42 paraffin-embedded nasopharyngeal carcinoma tissues were performed by immunohistochemistry. MTT assay, flow cytometry, and scratch tests were performed after CNE-2 cells were transfected with the best serum- and glucocorticoid-inducible kinase 3 shRNA plasmid selected by western blotting using lipofectamine to study its effect on cell proliferation, apoptosis, and migration. RESULTS: Serum- and glucocorticoid-inducible kinase 3 was overexpressed in human nasopharyngeal carcinoma tissues and cells. Serum- and glucocorticoid-inducible kinase 3 expression decreased markedly after CNE-2 cells were transfected with the serum- and glucocorticoid-inducible kinase 3 shRNA, leading to strong inhibition of cell proliferation and migration. In addition, the apoptosis rate increased in CNE-2 cells after serum- and glucocorticoid-inducible kinase 3 knockdown. CONCLUSION: Serum- and glucocorticoid-inducible kinase 3 expression was more frequently observed as the nasopharyngeal epithelium progresses from normal tissue to carcinoma. This suggests that serum- and glucocorticoid-inducible kinase 3 contributes to the multistep process of NPC carcinogenesis. Serum- and glucocorticoid-inducible kinase 3 represents a target for nasopharyngeal carcinoma therapy, and a basis exists for the further investigation of this adjuvant treatment modality for nasopharyngeal carcinoma.

Asunto(s)

Proteínas Inmediatas-Precoces/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adulto , Apoptosis , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Proteínas Inmediatas-Precoces/farmacología , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/patología , Nasofaringitis/metabolismo , Nasofaringitis/patología , Proteínas Serina-Treonina Quinasas/farmacología , ARN Interferente Pequeño/metabolismo

RESUMEN

Damage observed in the hippocampus of the adult spontaneously hypertensive rat (SHR) resembles the neuropathology of mineralocorticoid-induced hypertension, supporting a similar endocrine dysfunction in both entities. In the present study, we tested the hypothesis that increased expression of the hippocampal mineralocorticoid receptor (MR) in SHR animals is associated with a prevalent expression of pro-inflammatory over anti-inflammatory factors. Accordingly, in the hippocampus, we measured mRNA expression and immunoreactivity of the MR and glucocorticoid receptor (GR) using a quantitative polymerase chain reaction and histochemistry. We also measured serum-glucocorticoid-activated kinase 1 (Sgk1 mRNA), the number and phenotype of Iba1+ microglia, as well as mRNA expression levels of the pro-inflammatory factors cyclo-oxygenase 2 (Cox2), Nlrp3 inflammasome and tumour necrosis factor α (Tnfα). Expression of anti-inflammatory transforming growth factor (Tgf)ß mRNA and the NADPH-diaphorase activity of nitric oxide synthase (NOS) were also determined. The results showed that, in the hippocampus of SHR rats, expression of MR and the number of immunoreactive MR/GR co-expressing cells were increased compared to Wistar-Kyoto control animals. Expression of Sgk1, Cox2, Nlrp3 and the number of ramified glia cells positive for Iba1+ were also increased, whereas Tgfß mRNA expression and the NADPH-diaphorase activity of NOS were decreased. We propose that, in the SHR hippocampus, increased MR expression causes a bias towards a pro-inflammatory phenotype characteristic for hypertensive encephalopathy.

Asunto(s)

Hipocampo/metabolismo , Inflamación/metabolismo , Neuronas/metabolismo , Receptores de Mineralocorticoides/metabolismo , Animales , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Masculino , Microglía/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

SMCTs move several important fuel molecules that are involved in lipid, carbohydrate, and amino acid metabolism, but their regulation has been poorly studied. Insulin controls the translocation of several solutes that are involved in energetic cellular metabolism, including glucose. We studied the effect of insulin on the function of human SMCT1 expressed in Xenopus oocytes. The addition of insulin reduced α-keto-isocaproate (KIC)-dependent 22Na+ uptake by 29%. Consistent with this result, the coinjection of SMCT1 with SGK1 cRNA decreased the KIC-dependent 22Na+ uptake by 34%. The reduction of SMCT1 activity by SGK1 depends on its kinase activity, and it was observed that the coinjection of SMCT1 with S442D-SGK1 (a constitutively active mutant) decreased the KIC-dependent 22Na+ uptake by 50%. In contrast, an SMCT1 coinjection with K127M-SGK1 (an inactive mutant) had no effect on the KIC-dependent Na+ uptake. The decreasing SMCT1 function by insulin or SGK1 was corroborated by measuring [1-14C]acetate uptake and the electric currents of SMCT1-injected oocytes. Previously, we found that SMCT2/Slc5a12-mRNA, but not SMCT1/Slc5a8-mRNA, is present in zebrafish pancreas (by in situ hybridization); however, SLC5a8 gene silencing was associated with the development of human pancreatic cancer. We confirmed that the mRNA and protein of both transporters were present in rat pancreas using RT-PCR with specific primers, Western blot analysis, and immunohistochemistry. Additionally, significant propionate-dependent 22Na+ uptake occurred in pancreatic islets and was reduced by insulin treatment. Our data indicate that human SMCT1 is regulated by insulin and SGK1 and that both SMCTs are present in the mammalian pancreas.

Asunto(s)

Proteínas Inmediatas-Precoces/metabolismo , Insulina/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Sodio/metabolismo , Animales , ADN Complementario/metabolismo , Humanos , Masculino , Oocitos/metabolismo , Páncreas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Xenopus laevis/metabolismo , Pez Cebra/metabolismo

RESUMEN

The aim of this study was to identify differentially expressed genes (DEGs) in renal medullary hypertension and reveal their pathogenic mechanisms. We downloaded the gene expression profile of GSE28360 from the Gene Expression Omnibus database. The profile included 14 samples (5 normal and 9 hypertension). The DEGs in normal and disease samples were distinguished with a false-discovery rate threshold of <0.05 and a fold-change value of >2 or <-2. We put the selected genes into the online program String 8.3 to obtain the protein-protein interaction network and selected the hub proteins. These hub proteins were then placed in the PANTHER database to determine hub protein-related pathways and explain their functions. Finally, we cleared up the single-nucleotide polymorphisms (SNPs) of the hub genes via combing with the National Center for Biotechnology SNP database. A total of 13 genes were identified as DEGs between normal and disease samples. Five selected hub proteins, B-cell translocation gene 2 (BTG2), FBJ murine osteosarcoma viral oncogene homolog (FOS), nuclear receptor subfamily 4, group A, member 1 (NR4A1), NR4A member 2 (NR4A2), and NR4A member 3 (NR4A3), were mainly related to angiogenesis and B-cell activation. After SNP analysis, 103, 103, 595, 150, and 493 SNPs were found to correspond to BTG2, FOS, NR4A1, NR4A2, and NR4A3, respectively. Our results suggest that pathways of angiogenesis and B-cell activation may involve in the progression of renal medulla hypertension.

Asunto(s)

Linfocitos B/citología , Hipertensión Renal/genética , Activación de Linfocitos , Inductores de la Angiogénesis/metabolismo , Presión Sanguínea/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Análisis por Micromatrices , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Transcriptoma , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The Saccharomyces cerevisiae ypk1 and ypk2 genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the Aspergillus nidulans YPK1 homologue, YpkA, representing the first filamentous fungal YPK1 homologue. Two conditional mutant strains were constructed by replacing the endogenous ypk1 promoter with two different regulatable promoters, alcA (from the alcohol dehydrogenase gene) and niiA (from the nitrate reductase gene). Both constructs confirmed that ypkA was an essential gene in A. nidulans. Repression of ypkA caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by ypkA repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The A. nidulans Pkh1 homologue pkhA was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of pkhA or epistatic to pkhA, rather, ypkA and pkhA are genetically independent or in parallel. BarA is a homologue of the yeast Lag1 acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When barA was absent, ypkA repression was lethal to the cell. Therefore, there appears to be a genetic interaction between ypkA, barA, and the sphingolipid synthesis. Transcriptional profiling of ypkA overexpression and down-regulation revealed several putative YpkA targets associated with the observed phenotypes.

Asunto(s)

Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Acilcoenzima A/metabolismo , Aspergillus nidulans/genética , Catálisis , Membrana Celular/metabolismo , Endocitosis , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Familia de Multigenes , Mutación , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Esfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esporas Fúngicas/metabolismo

RESUMEN

Using polymerase chain reaction, a 1050-bp sequence of the US1 gene was amplified from the pseudorabies virus (PRV) Becker strain genome; identification of the US1 gene was confirmed by further cloning and sequencing. Bioinformatics analysis indicated that the PRV US1 gene encodes a putative polypeptide with 349 amino acids. The encoded protein, designated PICP22, had a conserved Herpes_IE68 domain, which was found to be closely related with the herpes virus immediate early regulatory protein family and is highly conserved among the counterparts encoded by Herpes_IE68 genes. Multiple nucleic acid sequence and amino acid sequence alignments suggested that the product of PRV US1 has a relatively higher homology with ICP22-like proteins of genus Varicellovirus than with those of other genera of Alphaherpesvirinae. In addition, phylogenetic analysis showed that PRV US1 has a close evolutionary relationship with members of the genus Varicellovirus, especially Equid herpes virus 1 (EHV-1), EHV-4 and EHV-9. Antigen prediction indicated that several potential B-cell epitopes are located in PICP22. Also, subcellular localization analysis demonstrated that PICP22 is predominantly located in the cytoplasm, suggesting that it might function as a cytoplasmic-targeted protein.

Asunto(s)

ADN Viral/genética , Genes Virales , Herpesvirus Suido 1/genética , Proteínas Virales/genética , Alphaherpesvirinae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular/métodos , Biología Computacional/métodos , Epítopos de Linfocito B/genética , Genoma Viral/genética , Proteínas Inmediatas-Precoces/genética , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Ácido Nucleico , Varicellovirus/genética

RESUMEN

Immediate-early 3 (IE3) gene products are required to activate early (E)-stage gene expression of murine cytomegaloviruses (MCMV). The first early gene activated by IE3 is the M112-113 gene (also called E1), although a complete understanding of the activation mechanism is still lacking. In this paper, we identify a 10-bp cis-regulating motif upstream of the M112-113 TATA box as important for IE3 activation of M112-113 expression. Results from DNA affinity assays and chromatin immunoprecipitation assays show that the association of IE3 with the M112-113 gene promoter was eliminated by deletion of the 10-bp DNA sequence, now named IE3AM (for IE3 activating motif). In addition, IE3 interacts with TATA box binding protein (TBP), a core protein of TFIID (transcription initiation) complexes. Finally, we created an IE3AM-deleted MCMV (MCMVdIE3AM) using a bacterial artificial chromosome system. The mutant virus can still replicate in NIH 3T3 cells but at a significantly lower level. The defectiveness of the MCMVdIE3AM infection can be rescued in an M112-113-complemented cell line. Our results suggest that the interactions of IE3 with IE3AM and with TBP stabilize the TFIID complex at the M112-113 promoter such that M112-113 gene expression can be activated and/or enhanced.

Asunto(s)

Regulación Viral de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Muromegalovirus/fisiología , Regiones Promotoras Genéticas , Proteínas Virales/genética , Replicación Viral , Animales , Línea Celular , ADN Viral/genética , ADN Viral/metabolismo , Expresión Génica , Infecciones por Herpesviridae/virología , Ratones , Muromegalovirus/genética , Muromegalovirus/metabolismo , Células 3T3 NIH , Eliminación de Secuencia , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Proteínas Virales/metabolismo

RESUMEN

Members of the Herpesviridae family have been implicated in a number of tumours in humans. At least 75% of the human population has had contact with cytomegalovirus (HCMV). In this work, we screened 75 Brazilian glioma biopsies for the presence of HCMV DNA sequences. HCMV DNA was detected in 36% (27/75) of the biopsies. It is possible that HCMV could be a co-factor in the evolution of brain tumours.

Asunto(s)

Neoplasias Encefálicas/virología , Infecciones por Citomegalovirus/complicaciones , Citomegalovirus/genética , ADN Viral/análisis , Glioma/virología , Adulto , Biopsia , Niño , Estudios de Cohortes , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/diagnóstico , Femenino , Humanos , Proteínas Inmediatas-Precoces/análisis , Proteínas Inmediatas-Precoces/inmunología , Masculino , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Prevalencia , Adulto Joven

RESUMEN

Members of the Herpesviridae family have been implicated in a number of tumours in humans. At least 75% of the human population has had contact with cytomegalovirus (HCMV). In this work, we screened 75 Brazilian glioma biopsies for the presence of HCMV DNA sequences. HCMV DNA was detected in 36% (27/75) of the biopsies. It is possible that HCMV could be a co-factor in the evolution of brain tumours.

Asunto(s)

Adulto , Niño , Femenino , Humanos , Masculino , Adulto Joven , Neoplasias Encefálicas/virología , Infecciones por Citomegalovirus/complicaciones , Citomegalovirus/genética , ADN Viral/análisis , Glioma/virología , Biopsia , Estudios de Cohortes , Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/inmunología , Proteínas Inmediatas-Precoces/análisis , Proteínas Inmediatas-Precoces/inmunología , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Prevalencia