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Martin R.,Institute Biologia y Genetica Molecular IBGM | Cordova C.,Institute Biologia y Genetica Molecular IBGM | Gutierrez B.,Institute Biologia y Genetica Molecular IBGM | Hernandez M.,Institute Biologia y Genetica Molecular IBGM | Nieto M.L.,Institute Biologia y Genetica Molecular IBGM
PLoS ONE | Year: 2017

Glioblastoma, the most aggressive type of primary brain tumour, shows worse prognosis linked to diabetes or obesity persistence. These pathologies are chronic inflammatory conditions characterized by altered profiles of inflammatory mediators, including leptin and secreted phospholipase A2-IIA (sPLA2-IIA). Both proteins, in turn, display diverse pro-cancer properties in different cell types, including astrocytes. Herein, to understand the underlying relationship between obesity and brain tumors, we investigated the effect of leptin, alone or in combination with sPLA2-IIA on astrocytoma cell functions. sPLA2-IIA induced up-regulation of leptin receptors in 1321N1 human astrocytoma cells. Leptin, as well as sPLA2-IIA, increased growth and migration in these cells, through activation/phosphorylation of key proteins of survival cascades. Leptin, at concentrations with minimal or no activating effects on astrocytoma cells, enhanced growth and migration promoted by low doses of sPLA2-IIA. sPLA2-IIA alone induced a transient phosphorylation pattern in the Src/ERK/Akt/mTOR/ p70S6K/rS6 pathway through EGFR transactivation, and co-addition of leptin resulted in a sustained phosphorylation of these signaling regulators. Mechanistically, EGFR transactivation and tyrosine- and serine/threonine-protein phosphatases revealed a key role in this leptin- sPLA2-IIA cross-talk. This cooperative partnership between both proteins was also found in primary astrocytes. These findings thus indicate that the adipokine leptin, by increasing the susceptibility of cells to inflammatory mediators, could contribute to worsen the prognosis of tumoral and neurodegenerative processes, being a potential mediator of some obesity- related medical complications. © 2017 Martín et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Senovilla L.,Institute Biologia y Genetica Molecular IBGM | Nunez L.,Institute Biologia y Genetica Molecular IBGM | Nunez L.,University of Valladolid | de Campos J.M.,Hospital Universitario Del Rio Hortega | And 5 more authors.
Frontiers in Oncology | Year: 2015

Human pituitary tumors are generally benign adenomas causing considerable morbidity due to excess hormone secretion, hypopituitarism, and other tumor mass effects. Pituitary tumors are highly heterogeneous and difficult to type, often containing mixed cell phenotypes. We have used calcium imaging followed by multiple immunocytochemistry to type growth hormone secreting (GHomas) and non-functioning pituitary adenomas (NFPAs). Individual cells were typed for stored hormones and calcium responses to classic hypothalamic releasing hormones (HRHs). We found that GHomas contained growth hormone cells either lacking responses to HRHs or responding to all four HRHs. However, most GHoma cells were polyhormonal cells responsive to both thyrotropin-releasing hormone (TRH) and GH-releasing hormone. NFPAs were also highly heterogeneous. Some of them contained ACTH cells lacking responses to HRHs or polyhormonal gonadotropes responsive to LHRH and TRH. However, most NFPAs were made of cells storing no hormone and responded only to TRH. These results may provide new insights on the ontogeny of GHomas and NFPAs. © 2015 Senovilla, Núñez, de Campos, de Luis, Romero, García-Sancho and Villalobos.


PubMed | Institute Biologia y Genetica Molecular IBGM, University of Valladolid and Hospital Universitario Del Rio Hortega
Type: | Journal: Frontiers in oncology | Year: 2015

Human pituitary tumors are generally benign adenomas causing considerable morbidity due to excess hormone secretion, hypopituitarism, and other tumor mass effects. Pituitary tumors are highly heterogeneous and difficult to type, often containing mixed cell phenotypes. We have used calcium imaging followed by multiple immunocytochemistry to type growth hormone secreting (GHomas) and non-functioning pituitary adenomas (NFPAs). Individual cells were typed for stored hormones and calcium responses to classic hypothalamic releasing hormones (HRHs). We found that GHomas contained growth hormone cells either lacking responses to HRHs or responding to all four HRHs. However, most GHoma cells were polyhormonal cells responsive to both thyrotropin-releasing hormone (TRH) and GH-releasing hormone. NFPAs were also highly heterogeneous. Some of them contained ACTH cells lacking responses to HRHs or polyhormonal gonadotropes responsive to LHRH and TRH. However, most NFPAs were made of cells storing no hormone and responded only to TRH. These results may provide new insights on the ontogeny of GHomas and NFPAs.


Santodomingo J.,Institute Biologia y Genetica Molecular IBGM | Santodomingo J.,University of Geneva | Fonteriz R.I.,Institute Biologia y Genetica Molecular IBGM | Lobaton C.D.,Institute Biologia y Genetica Molecular IBGM | And 3 more authors.
Cellular and Molecular Neurobiology | Year: 2010

We have investigated the dynamics of the free [Ca2+] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca 2+-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca2+] ([Ca2+] SG] was around 20-40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca2+ pumps with thapsigargin largely blocked Ca2+ uptake by the granules in Ca2+-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca2+ pump inhibitor benzohydroquinone induced a rapid release of Ca2+ from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca2+ pumps is essential to maintain the steady-state [Ca2+]SG. Both inositol 1,4,5-trisphosphate (InsP3) and caffeine produced a rapid Ca2+ release from the granules, suggesting the presence of InsP 3 and ryanodine receptors in the granules. The response to high-K+ depolarization was different in both cell types, a decrease in [Ca2+]SG in PC12 cells and an increase in [Ca 2+]SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca 2+]SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca2+ through SERCA-type Ca2+ pumps and can release it through InsP3 and ryanodine receptors, supporting the hypothesis that secretory granule Ca 2+ may be released during cell stimulation and contribute to secretion. © 2010 Springer Science+Business Media, LLC.


Barrio-Real L.,University of Salamanca | Barrueco M.,University of Salamanca | Gonzalez-Sarmiento R.,University of Salamanca | Caloca M.J.,Institute Biologia y Genetica Molecular IBGM
Journal of Investigative Medicine | Year: 2013

Objective: The CHN2 gene encodes the A2-chimaerin, a Rac-specific guanosine-5¶-triphosphatase activating protein with an important role in the establishment of functional brain circuitry by controlling axon pruning. Genetic studies suggest that the CHN2 gene harbors variants that contribute to addiction vulnerability and smoking behavior. To further evaluate the role of A2-chimaerin in nicotine addiction, we investigated the association of 3 individual polymorphisms of the CHN2 gene with smoking dependence. Methods: Three hundred sixty-one healthy volunteers, 173 smokers (mean T SD age, 60.4 T 1.4 years) and 188 control subjects (mean T SD age, 45.9 T 1.4 years) were genotyped for 3 single-nucleotide polymorphisms in the CHN2 gene (rs3750103, rs12112301, and rs186911567). The association of these polymorphisms with smoking habits was analyzed. Results: There was no significant association of polymorphisms rs12112301 and rs3750103 with smoking. However, there was a significant difference in the frequency of the rs186911567 polymorphism between the smokers and the controls (P = 0.003). Conclusions: We report for the first time a significant association of the novel rs186911567 polymorphism of the CHN2 gene with smoking. © 2013 by The American Federation for Medical Research.


Calvo-Rodriguez M.,Institute Biologia y Genetica Molecular IBGM | Calvo-Rodriguez M.,Harvard University | Garcia-Durillo M.,Institute Biologia y Genetica Molecular IBGM | Villalobos C.,Institute Biologia y Genetica Molecular IBGM | And 2 more authors.
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2016

Aging is associated to cognitive decline and susceptibility to neuron death, two processes related recently to subcellular Ca2+ homeostasis. Memory storage relies on mushroom spines stability that depends on store-operated Ca2 + entry (SOCE). In addition, Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria sustains energy production but mitochondrial Ca2+ overload promotes apoptosis. We have addressed whether SOCE and ER-mitochondria Ca2+ transfer are influenced by culture time in long-term cultures of rat hippocampal neurons, a model of neuronal aging. We found that short-term cultured neurons show large SOCE, low Ca2+ store content and no functional coupling between ER and mitochondria. In contrast, in long-term cultures reflecting aging neurons, SOCE is essentially lost, Stim1 and Orai1 are downregulated, Ca2+ stores become overloaded, Ca2+ release is enhanced, expression of the mitochondrial Ca2+ uniporter (MCU) increases and most Ca2 + released from the ER is transferred to mitochondria. These results suggest that neuronal aging is associated to increased ER-mitochondrial cross talking and loss of SOCE. This subcellular Ca2+ remodeling might contribute to cognitive decline and susceptibility to neuron cell death in the elderly. © 2016


Calvo M.,University of Valladolid | Sanz-Blasco S.,University of Valladolid | Caballero E.,University of Valladolid | Villalobos C.,University of Valladolid | And 2 more authors.
Journal of Neurochemistry | Year: 2015

Brain damage after insult and cognitive decline are related to excitotoxicity and strongly influenced by aging, yet mechanisms of aging-dependent susceptibility to excitotoxicity are poorly known. Several non-steroidal anti-inflammatory drugs (NSAIDs) may prevent excitotoxicity and cognitive decline in the elderly by an unknown mechanism. Interestingly, after several weeks in vitro, hippocampal neurons display important hallmarks of neuronal aging in vivo. Accordingly, rat hippocampal neurons cultured for several weeks were used to investigate mechanisms of aging-related susceptibility to excitotoxicity and neuroprotection by NSAIDs. We found that NMDA increased cytosolic Ca2+ concentration in young, mature and aged neurons but only promoted apoptosis in aged neurons. Resting Ca2+ levels and responses to NMDA increased with time in culture which correlated with changes in expression of NMDA receptor subunits. In addition, NMDA promoted mitochondrial Ca2+ uptake only in aged cultures. Consistently, specific inhibition of mitochondrial Ca2+ uptake decreased apoptosis. Finally, we found that a series of NSAIDs depolarized mitochondria and inhibited mitochondrial Ca2+ overload, thus preventing NMDA-induced apoptosis in aged cultures. We conclude that mitochondrial Ca2+ uptake is critical for age-related susceptibility to excitotoxicity and neuroprotection by NSAIDs. © 2014 International Society for Neurochemistry.


PubMed | Institute Biologia y Genetica Molecular IBGM
Type: | Journal: Journal of neuroinflammation | Year: 2012

Activation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimers disease (AD) and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA), an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells.Primary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events.The direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNF. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF) ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478), a matrix metalloproteinase inhibitor (GM6001), an ADAM inhibitor (TAPI-1), and a HB-EGF neutralizing antibody abrogated the phenotype of activated microglia induced by the sPLA2-IIA.These results support the hypothesis that sPLA2-IIA may act as a potent modulator of microglial functions through its ability to induce EGFR transactivation and HB-EGF release. Accordingly, pharmacological modulation of EGFR might be a useful tool for treating neuroinflammatory diseases characterized by sPLA2-IIA accumulation.


PubMed | Institute Biologia y Genetica Molecular IBGM and University of Valladolid
Type: Journal Article | Journal: Biochimica et biophysica acta | Year: 2016

Aging is associated to cognitive decline and susceptibility to neuron death, two processes related recently to subcellular Ca

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