Person: Benito De Las Heras, Manuel R.
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First Name
Manuel R.
Last Name
Benito De Las Heras
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Farmacia
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
6 results
Search Results
Now showing 1 - 6 of 6
Item Poly (ADP-ribose) polymerase 3 (PARP3), a potential repressor of telomerase activity(Journal of Experimental & Clinical Cancer Research, 2014) Fernández-Marcelo, Tamara; Frías, Cristina; Pascua, Irene; Juan Chocano, María Del Carmen De; Head, Jacqueline; Gómez, Ana; Hernando Trancho, Florentino; Jarabo Sarceda, José Ramón; Díaz-Rubio García, Eduardo; Torres García, Antonio José; Rouleau, Michèle; Benito De Las Heras, Manuel R.; Iniesta, Pilar; Iniesta Serrano, María PilarBackground Considering previous result in Non-Small Cell Lung Cancer (NSCLC), we investigated in human cancer cells the role of PARP3 in the regulation of telomerase activity. Methods We selected A549 (lung adenocarcinoma cell line) and Saos-2 (osteosarcoma cell line), with high and low telomerase activity levels, respectively. The first one was transfected using a plasmid construction containing a PARP3 sequence, whereas the Saos-2 cells were submitted to shRNA transfection to get PARP3 depletion. PARP3 expression on both cell systems was evaluated by real-time quantitative PCR and PARP3 protein levels, by Western-blot. Telomerase activity was determined by TRAP assay. Results In A549 cells, after PARP3 transient transfection, data obtained indicated that twenty-four hours after transfection, up to 100-fold increased gene expression levels were found in the transfected cells with pcDNA/GW-53/PARP3 in comparison to transfected cells with the empty vector. Moreover, 48 hours post-transfection, telomerase activity decreased around 33%, and around 27%, 96 hours post-transfection. Telomerase activity average ratio was 0.67 ± 0.05, and 0.73 ± 0.06, respectively, with significant differences. In Saos-2 cells, after shRNA-mediated PARP3 silencing, a 2.3-fold increase in telomerase activity was detected in relation to the control. Conclusion Our data indicated that, at least in some cancer cells, repression of PARP3 could be responsible for an increased telomerase activity, this fact contributing to telomere maintenance and, therefore, avoiding genome instability.Item Severe Hepatic Insulin Resistance Induces Vascular Dysfunction: Improvement by Liver-Specific Insulin Receptor Isoform A Gene Therapy in a Murine Diabetic Model(Cells, 2021) Gómez Hernández, María De La Almudena; Heras Jiménez, Natalia De Las; López-Pastor, Andrea R.; García-Gómez, Gema; Infante-Menéndez, Jorge; González-López, Paula; González-Illanes, Tamara; Lahera Julia, Vicente; Benito De Las Heras, Manuel R.; Escribano Illanes, ÓscarBackground: Cardiovascular dysfunction is linked to insulin-resistant states. In this paper, we analyzed whether the severe hepatic insulin resistance of an inducible liver-specific insulin receptor knockout (iLIRKO) might generate vascular insulin resistance and dysfunction, and whether insulin receptor (IR) isoforms gene therapy might revert it. Methods: We studied in vivo insulin signaling in aorta artery and heart from iLIRKO. Vascular reactivity and the mRNA levels of genes involved in vascular dysfunction were analyzed in thoracic aorta rings by qRT-PCR. Finally, iLIRKO mice were treated with hepatic-specific gene therapy to analyze vascular dysfunction improvement. Results: Our results suggest that severe hepatic insulin resistance was expanded to cardiovascular tissues. This vascular insulin resistance observed in aorta artery from iLIRKO mice correlated with a reduction in both PI3K/AKT/eNOS and p42/44 MAPK pathways, and it might be implicated in their vascular alterations characterized by endothelial dysfunction, hypercontractility and eNOS/iNOS levels’ imbalance. Finally, regarding long-term hepatic expression of IR isoforms, IRA was more efficient than IRB in the improvement of vascular dysfunction observed in iLIRKO mice. Conclusion: Severe hepatic insulin resistance is sufficient to produce cardiovascular insulin resistance and dysfunction. Long-term hepatic expression of IRA restored the vascular damage observed in iLIRKO mice.Item Specific knockout of p85α in brown adipose tissue induces resistance to high-fat diet–induced obesity and its metabolic complications in male mice(Molecular metabolism, 2020) Gómez Hernández, María De La Almudena; Raposo López-Pastor, Andrea; Rubio-Longas, Carlota; Majewski, Patrik; Beneit, Nuria; Viana-Huete, Vanesa; García-Gómez, Gema; Fernandez, Silvia; Hribal, Marta Letizia; Sesti, Giorgio; Escribano Illanes, Óscar; Benito De Las Heras, Manuel R.Objective: An increase in mass and/or brown adipose tissue (BAT) functionality leads to an increase in energy expenditure, which may be beneficial for the prevention and treatment of obesity. Moreover, distinct class I PI3K isoforms can participate in metabolic control as well as in systemic dysfunctions associated with obesity. In this regard, we analyzed in vivo whether the lack of p85a in BAT (BATp85aKO) could modulate the activity and insulin signaling of this tissue, thereby improving diet-induced obesity and its associated metabolic complications. Methods: We generated BATp85aKO mice using Cre-LoxP technology, specifically deleting p85a in a conditional manner. To characterize this new mouse model, we used mice of 6 and 12 months of age. In addition, BATp85aKO mice were submitted to a high-fat diet (HFD) to challenge BAT functionality. Results: Our results suggest that the loss of p85a in BAT improves its thermogenic functionality, high-fat dieteinduced adiposity and body weight, insulin resistance, and liver steatosis. The potential mechanisms involved in the improvement of obesity include (1) increased insulin signaling and lower activation of JNK in BAT, (2) enhanced insulin receptor isoform B (IRB) expression and association with IRS-1 in BAT, (3) lower production of proinflammatory cytokines by the adipose organ, (4) increased iWAT browning, and (5) improved liver steatosis. Conclusions: Our results provide new mechanisms involved in the resistance to obesity development, supporting the hypothesis that the gain of BAT activity induced by the lack of p85a has a direct impact on the prevention of diet-induced obesity and its associated metabolic complicationsItem Negative regulation of Akt activity by p38α MAP kinase in cardiomyocytes involves membrane localization of PP2A through interaction with caveolin-1(Cellular Signalling, 2007) Zuluaga, Susana; Álvarez-Barrientos, Alberto; Gutiérrez Uzquiza, Álvaro; Benito De Las Heras, Manuel R.; Nebreda, Angel R.; Porras, Almudena; Porras Gallo, María AlmudenaCardiomyocyte-derived cell lines deficient in p38α are more resistant to apoptosis owing to lower expression of the pro-apoptotic proteins Bax and Fas and upregulation of the ERK survival pathway. Here, we show that increased Akt activity also contributes to the enhanced survival of p38α-deficient cardiomyocytes. We found that the serine/threonine phosphatase PP2A can be targeted to caveolae through interaction with caveolin-1 in a p38α-dependent manner. In agreement with this, PP2A activity associated with caveolin-1 was higher in wild type than in p38α-deficient cells. Akt was also present in caveolae and incubation of wild-type cells with the PP2A inhibitor okadaic acid increases the levels of Akt activity. Thus, p38α-induced re-localization of PP2A to caveolae can lead to dephosphorylation and inhibition of Akt, which in turn would contribute to the decreased survival observed in wild type cells. However, cell detachment impairs the formation of the PP2A/caveolin-1 complex and, as a consequence, phospho-Akt levels and survival are no longer regulated by p38α in detached wild type cardiomyocytes. Our results suggest that p38α can negatively modulate Akt activity, independently of PI3K, by regulating the interaction between caveolin-1 and PP2A through a mechanism dependent on cell attachment.Item p38alpha MAPK can positively or negatively regulate Rac-1 activity depending on the presence of serum(FEBS Letters, 2007) Zuluaga, Susana; Gutiérrez Uzquiza, Álvaro; Bragado, Paloma; Alvarez-Barrientos, Alberto; Benito De Las Heras, Manuel R.; Nebreda, Angel R; Porras Gallo, María AlmudenaThe small GTP-ase Rac-1 can trigger p38 MAPK activation and, in turn, p38alpha can regulate signalling pathways that potentially impinge on Rac-1 activity. We have investigated the cross-talk between p38alpha and Rac-1 and found that p38alpha regulates the association between Rac-1 and caveolin-1 in serum-deprived cardiomyocytes. This interaction depends on cell attachment and correlates with higher levels of active Rac-1. Actin organization might regulate the formation of Rac-1-caveolin-1 complexes. In contrast, the Rac-1-caveolin-1 interaction is almost undetectable in the presence of serum, where Rac-1 activity is negatively regulated by p38alpha. Our results indicate that p38alpha can differentially contribute to Rac-1 activation depending on the presence of serum.Item C3G down-regulates p38 MAPK activity in response to stress by Rap-1 independent mechanisms: Involvement in cell death(Cellular Signalling, 2010) Gutiérrez Uzquiza, Álvaro; Arechederra, María; Molina, Isabel; Baños, Rocío; Maia, Vera; Benito De Las Heras, Manuel R.; Guerrero, Carmen; Porras Gallo, María AlmudenaWe present here evidences supporting a negative regulation of p38α MAPK activity by C3G in MEFs triggered by stress, which can mediate cell death or survival depending on the stimuli. Upon serum deprivation, C3G induces survival through inhibition of p38α activation, which mediates apoptosis. In contrast, in response to H2O2, C3G behaves as a pro-apoptotic molecule, as its knock-down or knock-out enhances survival through up-regulation of p38α activation, which plays an anti-apoptotic role under these conditions. Moreover, the C3G target, Rap-1, plays an opposite role, also through regulation of p38α MAPK activity. Our data also suggest that changes in the protein levels of some members of the Bcl-2 family could account for the regulation of cell death by C3G and/or Rap-1 through p38α MAPK. Bim/Bcl-xL ratio appears to be important in the regulation of cell survival, both upon serum deprivation and in response to H2O2. In addition, the increase in BNIP-3 levels induced by C3G knock-down in wt cells treated with H2O2 might play a role preventing cell death. Therefore, we can conclude that C3G is a negative regulator of p38α MAPK in MEFs, while Rap-1 is a positive regulator, but both, through the regulation of p38α activity, can promote cell survival or cell death depending on the stimuli.