Implicación de la quinasa humana VRK1 y el factor de transcripción SOX2 en la regulación del ciclo celular a través de la inducción de ciclina D1

Trabajo Fin de Máster, Máster en Biología y Clínica del Cáncer (2014-2015)Una correcta regulación del ciclo celular es necesaria para que el proceso de proliferación ocurra de forma adecuada. En este trabajo, demostramos que la quinasa VRK1 y el factor de transcripción SOX2 regulan la progresión del ciclo celular induciendo la expresión de ciclina D1(CCND1), proteína implicada en la transición G1/S. Por el contrario, las macrohistonas mH2A1.2 y mH2A2 actúan como represores transcripcionales, inhibiendo la expresión de VRK1 al impedir el acceso del factor de transcripción SOX2 a las regiones promotoras de sus genes diana. Alteraciones de los niveles de SOX2, VRK1 y macrohistonas provocan la desregulación de los procesos de proliferación y diferenciación pudiendo causar tumorigénesis. Teniendo en cuenta estos resultados, se propone la necesidad de profundizar en el estudio de estas macrohistonas junto a VRK1 y SOX2 considerándolas como posibles candidatas para el desarrollo de nuevas terapias contra el cáncer.Peer Reviewe

Rol of lipotoxicity and mithocondrial dysfunction in cardiac and metabolic alterations associated with obesity

Tesis de la Universidad Complutense de Madrid, Facultad de Medicina, Departamento de Fisiología, leída el 20-11-2018La obesidad se define como una excesiva acumulación de grasa debido a undesequilibrio entre las calorías consumidas y gastadas. La obesidad mundial ha crecido dramáticamente en las últimas décadas por lo que no es sorprendente que numerosos estudios se hayan llevado a cabo para investigar su efecto dañino sobre la salud humana.Una de las características de los pacientes obesos es la presencia de adipocitos más grandes los cuáles una vez alcanzado su límite de expansión tienen una menor capacidad de almacenamiento de grasa dando lugar a un aumento de los niveles de ácidos grasos circulantes los cuales se pueden depositar en tejidos no adiposos, incluido el corazón, ejerciendo efectos tóxicos en los mismos. Además, la obesidad se asocia con alteraciones estructurales y funcionales en el corazón las cuales pueden dar lugar a disfunción cardiaca. De hecho, las enfermedades cardiovasculares son la primera causa de muerte en individuos con obesidad o diabetes. Entre los mecanismos que contribuyen a la aparición de alteraciones estructurales y funcionales se encuentran la lipotoxicidad cardiaca, las alteraciones en el uso de sustratos metabólicos, mediadores profibróticos como Galectina 3 (Gal-3), la disfunción mitocondrial y el estrés oxidativo, los cuales facilitan el desarrollo de fibrosis cardiaca con consecuencias funcionales. Niveles patológicos de especies reactivas de oxígeno siendo la mitocondria la principal fuente de los mismos, se ha demostrado que tienen un efecto dañino en el corazón...Obesity is defined as an abnormal or excessive fat accumulation due to an energy imbalance between calories consumed and expended. Worldwide obesity has grown dramatically in the last decades so it is not surprising that numerous studies have been performed to investigate its harmful effect on human health. One of the characteristics of obese patients is the presence of larger adipocytes which reach a limit and have diminished capacity to store fat, thereby leading to elevation of circulating fatty acids which can deposit in non-adipose tissues, including the heart,exerting toxic effects. Moreover, obesity is associated with structural and functional alterations in the heart that can lead to cardiac dysfunction. In fact, cardiovascular disease is the primary cause of death in individuals with obesity and diabetes. Among the mechanisms that contribute to cardiac structural and functional alterations, cardiac lipotoxicity, alterations in the use of metabolic substrates, profibrotic mediators such as Galectin-3 (Gal-3), mitochondrial dysfunction and oxidative stress are some of these which facilitate the development of cardiac fibrosis with functional consequences. Pathological ROS levels have been demonstrated to have harmful effects in the heart, with the mitochondria being the main source of ROS. Moreover, oxidative stress can also participate in the adipose tissue remodelling which can be underlying metabolic alterations associated with obesity...Depto. de FisiologíaFac. de MedicinaTRUEunpu

Lipotoxicity and Diabetic Nephropathy: Novel Mechanistic Insights and Therapeutic Opportunities

Lipotoxicity is characterized by the ectopic accumulation of lipids in organs different from adipose tissue. Lipotoxicity is mainly associated with dysfunctional signaling and insulin resistance response in non-adipose tissue such as myocardium, pancreas, skeletal muscle, liver, and kidney. Serum lipid abnormalities and renal ectopic lipid accumulation have been associated with the development of kidney diseases, in particular diabetic nephropathy. Chronic hyperinsulinemia, often seen in type 2 diabetes, plays a crucial role in blood and liver lipid metabolism abnormalities, thus resulting in increased non-esterified fatty acids (NEFA). Excessive lipid accumulation alters cellular homeostasis and activates lipogenic and glycogenic cell-signaling pathways. Recent evidences indicate that both quantity and quality of lipids are involved in renal damage associated to lipotoxicity by activating inflammation, oxidative stress, mitochondrial dysfunction, and cell-death. The pathological effects of lipotoxicity have been observed in renal cells, thus promoting podocyte injury, tubular damage, mesangial proliferation, endothelial activation, and formation of macrophage-derived foam cells. Therefore, this review examines the recent preclinical and clinical research about the potentially harmful effects of lipids in the kidney, metabolic markers associated with these mechanisms, major signaling pathways affected, the causes of excessive lipid accumulation, and the types of lipids involved, as well as offers a comprehensive update of therapeutic strategies targeting lipotoxicity

The coming age of flavonoids in the treatment of diabetic complications

Diabetes mellitus (DM), and its micro and macrovascular complications, is one of the biggest challenges for world public health. Despite overall improvement in prevention, diagnosis and treatment, its incidence is expected to continue increasing over the next years. Nowadays, finding therapies to prevent or retard the progression of diabetic complications remains an unmet need due to the complexity of mechanisms involved, which include inflammation, oxidative stress and angiogenesis, among others. Flavonoids are natural antioxidant compounds that have been shown to possess anti-diabetic properties. Moreover, increasing scientific evidence has demonstrated their potential anti-inflammatory and anti-oxidant effects. Consequently, the use of these compounds as anti-diabetic drugs has generated growing interest, as is reflected in the numerous in vitro and in vivo studies related to this field. Therefore, the aim of this review is to assess the recent pre-clinical and clinical research about the potential effect of flavonoids in the amelioration of diabetic complications. In brief, we provide updated information concerning the discrepancy between the numerous experimental studies supporting the eficacy of flavonoids on diabetic complications and the lack of appropriate and well-designed clinical trials. Due to the well-described beneficial effects on different mechanisms involved in diabetic complications, the excellent tolerability and low cost, future randomized controlled studies with compounds that have adequate bioavailability should be evaluated as add-on therapy on well-established anti-diabetic drugsThis paper was not funded. The authors work has been supported by FEDER-ISCIII Funds (PI17/00130, PI17/01495), Spanish Ministry of Economy and Competitiveness (RTI2018-098788-B-100, DTS17/00203, DTS19/00093, RYC-2017-22369), Spanish Society of Cardiology (SEC), Spanish Society of Nephrology (SEN) and Spanish Society of Atherosclerosis (SEA). TCO is an employee of FAES Pharma. The authors (except JAM) have an ongoing research project in common with FAES Pharma on Flavonoids in diabetic complications under the auspices of the joint-RETOS Collaborations Project 2017 (RTC-2017-6089-1), program supported by Spanish Ministry of Economy and Competitiveness

Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation.

Sox2 is a pluripotency transcription factor that as an oncogene can also regulate cell proliferation. Therefore, genes implicated in several different aspects of cell proliferation, such as the VRK1 chromatin-kinase, are candidates to be targets of Sox2. Sox 2 and VRK1 colocalize in nuclei of proliferating cells forming a stable complex. Sox2 knockdown abrogates VRK1 gene expression. Depletion of either Sox2 or VRK1 caused a reduction of cell proliferation. Sox2 up-regulates VRK1 expression and both proteins cooperate in the activation of CCND1. The accumulation of VRK1 protein downregulates SOX2 expression and both proteins are lost in terminally differentiated cells. Induction of neural differentiation with retinoic acid resulted in downregulation of Sox2 and VRK1 that inversely correlated with the expression of differentiation markers such as N-cadherin, Pax6, mH2A1.2 and mH2A2. Differentiation-associated macro histones mH2A1.2and mH2A2 inhibit CCND1 and VRK1 expression and also block the activation of the VRK1 promoter by Sox2. VRK1 is a downstream target of Sox2 and both form an autoregulatory loop in epithelial cell differentiation.Ministerio de Economía y Competitividad [SAF2013-44810R, SAF2014-57791-REDC].Consejería de Educación de la Junta de Castilla y León [CSI002U14 and UIC-017] to P.A.L

Nephroprotective effects of synthetic flavonoid hidrosmin in experimental diabetic nephropathy

Diabetes mellitus (DM) is a high‐impact disease commonly characterized by hyperglycemia, inflammation, and oxidative stress. Diabetic nephropathy (DN) is a common diabetic microvascular complication and the leading cause of chronic kidney disease worldwide. This study investigates the protective effects of the synthetic flavonoid hidrosmin (5‐O‐(beta-hydroxyethyl) diosmin) in experimental DN induced by streptozotocin injection in apolipoprotein E deficient mice. Oral administration of hidrosmin (300 mg/kg/day, n = 11) to diabetic mice for 7 weeks markedly reduced albuminuria (albumin‐to‐creatinine ratio: 47 ± 11% vs. control) and ameliorated renal pathological damage and expression of kidney injury markers. Kidneys of hidrosmin‐treated mice exhibited lower content of macrophages and T cells, reduced expression of cytokines and chemokines, and attenuated inflammatory signaling pathways. Hidrosmin treatment improved the redox balance by reducing prooxidant enzymes and enhancing antioxidant genes, and also decreased senescence markers in diabetic kidneys. In vitro, hidrosmin dose‐dependently reduced the expression of inflammatory and oxidative genes in tubuloepithelial cells exposed to either high‐glucose or cytokines, with no evidence of cytotoxicity at effective concentrations. In conclusion, the synthetic flavonoid hidrosmin exerts a beneficial effect against DN by reducing inflammation, oxidative stress, and senescence pathways. Hidrosmin could have a potential role as a coadjutant therapy for the chronic complications of DM.This work was supported by grants from the Spanish Ministry of Science and Innovation- FEDER funds (Retos Colaboración RTC2017-6089-1 and Retos Investigación RTI2018-098788-B-I00) and Instituto de Salud Carlos III (PI20/00487 and DTS 19/00093

El impacto de la obesidad sobre el lipidoma cardíaco y sus consecuencias en el daño cardíaco en ratas obesas

To explore the impact of obesity on the cardiac lipid profile in rats with diet-induced obesity, as well as to evaluate whether or not the specific changes in lipid species are associated with cardiac fibrosis. Methods: Male Wistar rats were fed either a high-fat diet (HFD, 35% fat) or standard diet (3.5% fat) for 6 weeks. Cardiac lipids were analyzed using by liquid chromatography-tandem mass spectrometry. Results: HFD rats showed cardiac fibrosis and enhanced levels of cardiac superoxide anion (O 2 ), HOMA index, adiposity, and plasma leptin, as well as a reduction in those of cardiac glucose transporter (GLUT 4), compared with control animals. Cardiac lipid profile analysis showed a significant increase in triglycerides, especially those enriched with palmitic, stearic, and arachidonic acid. An increase in levels of diacylglycerol (DAG) was also observed. No changes in cardiac levels of diacyl phosphatidylcholine, or even a reduction in total levels of diacyl phosphatidylethanolamine, diacyl phosphatidylinositol, and sphingomyelins (SM) was observed in HFD, as compared with control animals. After adjustment for other variables (oxidative stress, HOMA, cardiac hypertrophy), total levels of DAG were independent predictors of cardiac fibrosis while the levels of total SM were independent predictors of the cardiac levels of GLUT 4. Conclusions: These data suggest that obesity has a significant impact on cardiac lipid composition, although it does not modulate the different species in a similar manner. Nonetheless, these changes are likely to participate in the cardiac damage in the context of obesity, since total DAG levels can facilitate the development of cardiac fibrosis, and SM levels predict GLUT4 levelsThis work was supported by funds from the Sociedad Española de Arteriosclerosis (Basic Research Award 2015), from Plan Estatal I+D+I 2013-2016: PI15/01060 and SAF2016-81063. The study was cofunded by Fondo Europeo de Desarrollo Regional (FEDER), a way to build Europ

SOX2 regulates proliferation through VRK1 promoter activation

Resumen del trabajo presentado al 15th ASEICA International Congress, celebrado en Sevilla (España) del 21 al 23 de octubre de 2015.[Introduction]: Cell proliferation and cell differentiation are two distinct processes occurring at different stages of the cell life. Nonetheless, the regulatory mechanisms involved in the switch from proliferation to differentiation and the role and fate of proliferation-related proteins are yet to be fully understood. Sox2, a member of the SRY-related HMG-box family of transcriptional factors, is a core regulator of cell pluripotency and cell proliferation, survival and differentiation. Moreover, Sox2 may act as a cancer initiating gene in poorly differentiated or undifferentiated tumors, since these types of cancers have been characterized by many phenotypic traits similar to undifferentiated embryonic stem cells. Sox2 activates the promoter of yclin D1. In turn, VRK1 (Vaccinia-related Kinase-1) is a serine-threonine kinase ubiquitously expressed, with higher expression in highly-proliferative tissues, such as tumours. Thus, based on VRK1 features and expression, this kinase was related with the regulation of cell cycle and cell proliferation. Nowadays, it is known that VRK1 takes part in the correct progression of cell cycle through transcriptional factors regulation, regulation of proliferative proteins like Cyclin D1, modulation of p53 levels, nuclear envelope assembly and chromatin condensation. [Objectives]: We hypothesize that VRK1 regulates and is regulated Sox2. This regulation is important to maintain cell proliferation. Besides, we hypothesize that VRK1 could act as a transcriptional partner for Sox2 on the activation of CCND1 gene.[Methods]: The levels and co-localization of VRK1 and Sox2 were analyzed by immunofluorescence (IF) and immunohistochemistry. Interaction between VRK1 and Sox2 was assessed by reciprocal immunoprecipitations in Ntera-2 cells and MDA-MB-231 cells. Moreover, the effect of cell differentiation on VRK1 and Sox2 levels was studied by qRT-PCR, Western blot and IF. The activation of VRK1 promoter by Sox2 overexpression was assessed by luciferase assays, qRT-PCR, Western blot and IF. Moreover, the cooperation between VRK1 and Sox2 on CCND1 gene activation was evaluated by luciferase assays. [Results]: Here, we showed that VRK1 and Sox2 interact and co-localize in Ntera-2 and MDA-MB-231 and, in undifferentiated layers of stratified squamous epithelium. Sox2 overexpression induces an increase in the VRK1 promoter activity, alongside with an increment on VRK RNA and protein levels. On the other hand, down-regulation of VRK1 leads to an increase in Sox2 RNA and protein levels, while the overexpression has the opposite effect. Also VRK1 and Sox2 can activate, by themselves and in cooperation, expression of the CCND1 (cyclinD1) gene. This effect of VRK1 and Sox2 is mediated by the proximal promoter of CCND1. After induction of cell differentiation with retinoic acid, both Sox2 and VRK1 are downregulated and they are absent in terminally differentiated epithelial cells. [Conclusions]: VRK1 regulates and is regulated by Sox2. Additionally, VRK1 and Sox2 cooperate on the activation of CCND1 gene.Peer Reviewe

Análisis del lipidoma cardiaco en ratas obesas: Consecuencia fisiopatológicas

Trabajo presentado al XXX Congreso Nacional de la Sociedad Española de Arteriosclerosis (SEA), celebrado en Cádiz del 31 de mayo al 2 de junio de 2017.Peer reviewe