165 results on '"Juan A, Rosado"'
Search Results
2. Store-Operated Calcium Entry and Its Implications in Cancer Stem Cells
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Isaac Jardin, Jose J. Lopez, Jose Sanchez-Collado, Luis J. Gomez, Gines M. Salido, and Juan A. Rosado
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ORAI1 Protein ,Neoplasms ,Neoplastic Stem Cells ,Humans ,Calcium ,General Medicine ,Calcium Channels ,Calcium Signaling - Abstract
Tumors are composed by a heterogeneous population of cells. Among them, a sub-population of cells, termed cancer stem cells, exhibit stemness features, such as self-renewal capabilities, disposition to differentiate to a more proliferative state, and chemotherapy resistance, processes that are all mediated by Ca2+. Ca2+ homeostasis is vital for several physiological processes, and alterations in the patterns of expressions of the proteins and molecules that modulate it have recently become a cancer hallmark. Store-operated Ca2+ entry is a major mechanism for Ca2+ entry from the extracellular medium in non-excitable cells that leads to increases in the cytosolic Ca2+ concentration required for several processes, including cancer stem cell properties. Here, we focus on the participation of STIM, Orai, and TRPC proteins, the store-operated Ca2+ entry key components, in cancer stem cell biology and tumorigenesis.
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- 2022
3. Functional role of TRPC6 and STIM2 in cytosolic and endoplasmic reticulum Ca2+ content in resting estrogen receptor-positive breast cancer cells
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Lucia Gonzalez-Gutierrez, Isaac Jardin, Ginés M. Salido, Juan A. Rosado, Jose J. Lopez, Carlos Cantonero, and Jose Sanchez-Collado
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Estrogen receptor ,Breast Neoplasms ,Endoplasmic Reticulum ,Biochemistry ,TRPC6 ,03 medical and health sciences ,0302 clinical medicine ,TRPC6 Cation Channel ,Humans ,Stromal Interaction Molecule 2 ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Chemistry ,Endoplasmic reticulum ,STIM1 ,Cell Biology ,Transfection ,STIM2 ,Neoplasm Proteins ,Cell biology ,Cytosol ,Receptors, Estrogen ,030220 oncology & carcinogenesis ,MCF-7 Cells ,Calcium ,Female ,Homeostasis - Abstract
TRPC6 forms non-selective cation channels activated by a variety of stimuli that are involved in a wide number of cellular functions. In estrogen receptor-positive (ER+) breast cancer cells, the store-operated Ca2+ entry has been reported to be dependent on STIM1, STIM2 and Orai3, with TRPC6 playing a key role in the activation of store-operated Ca2+ entry as well as in proliferation, migration and viability of breast cancer cells. We have used a combination of biotinylation, Ca2+ imaging as well as protein knockdown and overexpression of a dominant-negative TRPC6 mutant (TRPC6dn) to show that TRPC6 and STIM2 are required for the maintenance of cytosolic and endoplasmic reticulum Ca2+ content under resting conditions in ER+ breast cancer MCF7 cells. These cells exhibit a greater plasma membrane expression of TRPC6 under resting conditions than non-tumoral breast epithelial cells. Attenuation of STIM2, TRPC6 and Orai3, alone or in combination, results in impairment of resting cytosolic and endoplasmic reticulum Ca2+ homeostasis. Similar results were observed when cells were transfected with expression plasmid for TRPC6dn. TRPC6 co-immunoprecipitates with STIM2 in resting MCF7 cells, a process that is impaired by rises in cytosolic Ca2+ concentration. Impairment of TRPC6 function leads to abnormal Ca2+ homeostasis and endoplasmic reticulum stress, thus, suggesting that TRPC6 might be a potential target for the development of anti-tumoral therapies.
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- 2020
4. Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells
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Jose Sanchez-Collado, Jose J. Lopez, Isaac Jardin, Alejandro Berna-Erro, Pedro J. Camello, Carlos Cantonero, Tarik Smani, Gines M. Salido, Juan A. Rosado, Consejo Superior de Investigaciones Científicas (España), Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Junta de Extremadura, Ministerio de Ciencia, Innovación y Universidades (España), and Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica
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Pharmacology ,ORAI1 Protein ,TRPC1 ,STIM1 ,Cell Membrane ,Store-operated calcium entry ,Cell Biology ,Orai1α ,Orai1β ,Cellular and Molecular Neuroscience ,Cations ,Molecular Medicine ,Humans ,Original Article ,Calcium ,Mutant Proteins ,Stromal Interaction Molecule 1 ,Molecular Biology ,HeLa Cells ,Protein Binding ,TRPC Cation Channels - Abstract
The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Grants PID2019-104084 GB-C21 and PID2019-104084 GB-C22 funded by MCIN/AEI/ https://doi.org/10.13039/501100011033 and ERDF A way of making Europe, and Junta de Extremadura-Fondo Europeo de Desarrollo Regional (FEDER; Grants IB20007, IB18025, GR21008 and GR18061) to JAR and TS. JJL and IJ are supported by a contract from Junta de Extremadura (TA18011 and TA18054, respectively). JS-C is supported by a contract from Ministry of Science, Innovation, and Universities, Spain. CC is supported by a Predoctoral fellowship of the Junta de Extremadura (PD16072). AB-E is supported by Junta de Extremadura (DPCI0643).
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- 2022
5. Phytochemical, Anti-diabetic and Cardiovascular Properties of Urtica dioica L. (Urticaceae): A Review
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Juan A. Rosado and Mohammed El Haouari
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Drug ,media_common.quotation_subject ,Phytochemicals ,Anti-Inflammatory Agents ,Antioxidants ,Diabetes mellitus ,Drug Discovery ,medicine ,Animals ,Humans ,Hypoglycemic Agents ,Urtica dioica ,Adverse effect ,Antihypertensive Agents ,media_common ,Pharmacology ,Traditional medicine ,biology ,Mechanism (biology) ,business.industry ,Type 2 Diabetes Mellitus ,Cardiovascular Agents ,General Medicine ,medicine.disease ,biology.organism_classification ,Urticaceae ,Diabetes Mellitus, Type 2 ,Phytochemical ,Cardiovascular Diseases ,business ,Phytotherapy - Abstract
Type 2 diabetes mellitus and cardiovascular diseases (CVD) have become the main cause of morbidity and death worldwide. In addition, current anti-diabetic and cardiovascular therapy is based on conventional drugs that have limited effectiveness and adverse side effects. In this regard, the role of medicinal herbs as a complementary or an alternative medicine is of great interest. Urtica dioica L. (Urticaceae), which is the focus of this review, has been widely used in traditional medicine to treat a variety of ailments, including, diabetes, hypertension and prostate cancer. The aim of this article is to review current knowledge related to the anti-diabetic and cardiovascular properties of U. dioica, with particular emphasis on the bioactive compounds, the plant parts used, and the action mechanism behind lowering blood glucose level and reducing risk of CVD. We also discuss the chemical composition and toxicological properties of the plant. From this review, it was suggested that the anti-diabetic and the cardiovascular effects of U. dioica are attributed to different classes of compounds, such as polyphenols, triterpens, sterols, flavonoids, and lectin which reduce the blood glucose level and the risk of CVD by their antihypertensive, antioxidant and anti-inflammatory properties and/or by interfering with different cellular signalization pathways, including increase of NO, inhibition of α-amylase and α-glycosidase, modulation of GLUT4 and protection of pancreatic β-cells, among others. The identification of the plant constituents and the understanding of their exact action mechanisms are necessary to prove the efficacy of the plant and develop it as pharmacological drug.
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- 2018
6. Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells
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Tarik Smani, Raquel Diez-Bello, Ginés M. Salido, Isaac Jardin, Débora Falcón, Sandra Alvarado, S. Regodon, Juan A. Rosado, [Jardin, Isaac] Univ Extremadura, Inst Mol Pathol Biomarkers IMPB, Dept Physiol, Cellular Physiol Res Grp, Caceres, Spain, [Diez-Bello, Raquel] Univ Extremadura, Inst Mol Pathol Biomarkers IMPB, Dept Physiol, Cellular Physiol Res Grp, Caceres, Spain, [Alvarado, Sandra] Univ Extremadura, Inst Mol Pathol Biomarkers IMPB, Dept Physiol, Cellular Physiol Res Grp, Caceres, Spain, [Salido, Gines M.] Univ Extremadura, Inst Mol Pathol Biomarkers IMPB, Dept Physiol, Cellular Physiol Res Grp, Caceres, Spain, [Rosado, Juan A.] Univ Extremadura, Inst Mol Pathol Biomarkers IMPB, Dept Physiol, Cellular Physiol Res Grp, Caceres, Spain, [Falcon, Debora] Inst Biomed Sevilla, Cardiovasc Physiopathol Grp, Seville, Spain, [Regodon, Sergio] Univ Extremadura, Dept Anim Med, Caceres, Spain, [Smani, Tarik] Inst Biomed Sevilla, Dept Med Physiol & Biophys, Seville, Spain, MICINN, Junta de Extremadura-Fondo Europeo de Desarrollo Regional, Junta de Extremadura-FEDER, CIBERCV (ISCIII, Madrid), Ministerio de Ciencia e Innovación (España), European Commission, Junta de Extremadura, and Instituto de Salud Carlos III
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0301 basic medicine ,TRPC6 ,Expression ,Apoptosis ,Triple Negative Breast Neoplasms ,OAG, 1-oleoyl-2-acetyl-sn-glycerol ,Hippocampus ,Trpm2 ,Biochemistry ,Calcium entry ,Antioxidants ,Induced oxidative stress ,BrdU, bromodeoxyuridine ,Cell Movement ,HER2, human epidermal growth factor receptor 2 ,Melatonin ,Modulation ,Chemistry ,ORAI1 ,Store-operated calcium entry ,SOCE, store-operated Ca2+ entry ,hormones, hormone substitutes, and hormone antagonists ,Research Article ,SOCE ,medicine.drug ,Down-Regulation ,TNBC, triple-negative breast cancer ,Cell Line ,ER, endoplasmic reticulum ,PI, propidium iodide ,03 medical and health sciences ,Downregulation and upregulation ,TG, Thapsigargin ,Cell Line, Tumor ,Channels ,Triple-negative breast cancer cells ,medicine ,TRPC6 Cation Channel ,HBS, HEPES-buffered saline ,Humans ,Viability assay ,OASF, Orai1-activating small fragment ,Molecular Biology ,Cell Proliferation ,030102 biochemistry & molecular biology ,Cell growth ,Cell Biology ,030104 developmental biology ,Cell culture ,Cancer research ,BSA, bovine serum albumin ,Calcium Channels - Abstract
Melatonin has been reported to induce effective reduction in growth and development in a variety of tumors, including breast cancer. In triple-negative breast cancer (TNBC) cells, melatonin attenuates a variety of cancer features, such as tumor growth and apoptosis resistance, through a number of still poorly characterized mechanisms. One biological process that is important for TNBC cells is store-operated Ca2+ entry (SOCE), which is modulated by TRPC6 expression and function. We wondered whether melatonin might intersect with this pathway as part of its anticancer activity. We show that melatonin, in the nanomolar range, significantly attenuates TNBC MDA-MB-231 cell viability, proliferation, and migration in a time- and concentration-dependent manner, without having any effect on nontumoral breast epithelial MCF10A cells. Pretreatment with different concentrations of melatonin significantly reduced SOCE in MDA-MB-231 cells without altering Ca2+ release from the intracellular stores. By contrast, SOCE in MCF10A cells was unaffected by melatonin. In the TNBC MDA-MB-468 cell line, melatonin not only attenuated viability, migration, and SOCE, but also reduced TRPC6 expression in a time- and concentration-dependent manner, without altering expression or function of the Ca2+ channel Orai1. The expression of exogenous TRPC6 overcame the effect of melatonin on SOCE and cell proliferation, and silencing or inhibition of TRPC6 impaired the inhibitory effect of melatonin on SOCE. These findings indicate that TRPC6 downregulation might be involved in melatonin's inhibitory effects on Ca2+ influx and the maintenance of cancer hallmarks and point toward a novel antitumoral mechanism of melatonin in TNBC cells., Supported by MICINN (Grants BFU2016-74932-C2-1-P, BFU2016-74932-C2-2-P, PID2019-104084GB-C21 and PID2019-104084GB-C22) and Junta de Extremadura-Fondo Europeo de Desarrollo Regional (Grants IB16046 and GR18061). S. A., R. D.-B., and I. J. (contract TA18054) are supported by contracts from Junta de Extremadura-FEDER. D. F. is supported by Jordi Soler grant from CIBERCV (ISCIII, Madrid).
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- 2020
7. ELA/APELA precursor cleaved by furin displays tumor suppressor function in renal cell carcinoma through mTORC1 activation
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Abdel-Majid Khatib, Jean Descarpentrie, Malaurie Eugenie, Amandine Mouchard, Juan A. Rosado, Mercedes Tomé, Isabelle Soubeyran, Jean-Luc Hoepffner, Isabel D. Alves, Macha Nikolski, Jose J. Lopez, Raúl V. Durán, Serge Evrard, Marielle Dubreuil, Geraldine Siegfried, Bruno O. Villoutreix, Katarzyna B. Hooks, Clément Bodineau, Fabienne Soulet, Composantes innées de la réponse immunitaire et différenciation (CIRID), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Institut Européen de Chimie et Biologie (IECB), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), Laboratoire de pharmacologie expérimentale et clinique : cibles moléculaires en cancérologie ((U 716)), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Ligue Nationale contre le Cancer (France), Conseil régional d'Aquitaine, Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM), École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)
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0301 basic medicine ,Angiogenesis ,Peptide Hormones ,[SDV]Life Sciences [q-bio] ,Chromophobe Renal Cell Carcinoma ,mTORC1 ,Mechanistic Target of Rapamycin Complex 1 ,Biology ,Kidney ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Cell Movement ,Sunitinib ,medicine ,Animals ,Humans ,Carcinoma, Renal Cell ,Furin ,ComputingMilieux_MISCELLANEOUS ,Cell Proliferation ,Apelin receptor ,Apelin Receptors ,Cell growth ,Tumor Suppressor Proteins ,General Medicine ,3. Good health ,Apelin ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,Intercellular Signaling Peptides and Proteins ,Calcium ,Signal Transduction ,Research Article - Abstract
Apelin is a well-established mediator of survival and mitogenic signaling through the apelin receptor (Aplnr) and has been implicated in various cancers; however, little is known regarding Elabela (ELA/APELA) signaling, also mediated by Aplnr, and its role and the role of the conversion of its precursor proELA into mature ELA in cancer are unknown. Here, we identified a function of mTORC1 signaling as an essential mediator of ELA that repressed kidney tumor cell growth, migration, and survival. Moreover, sunitinib and ELA showed a synergistic effect in repressing tumor growth and angiogenesis in mice. The use of site-directed mutagenesis and pharmacological experiments provided evidence that the alteration of the cleavage site of proELA by furin induced improved ELA antitumorigenic activity. Finally, a cohort of tumors and public data sets revealed that ELA was only repressed in the main human kidney cancer subtypes, namely clear cell, papillary, and chromophobe renal cell carcinoma. Aplnr was expressed by various kidney cells, whereas ELA was generally expressed by epithelial cells. Collectively, these results showed the tumor-suppressive role of mTORC1 signaling mediated by ELA and established the potential use of ELA or derivatives in kidney cancer treatment., This work was supported by INSERM, SIRIC Brio, Region Nouvelle Aquitaine, and La Ligue Contre le Cancer.
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- 2020
8. TRPC6 channel and its implications in breast cancer: an overview
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Ginés M. Salido, Juan A. Rosado, Isaac Jardin, and Joel Nieto
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0301 basic medicine ,Subfamily ,Breast Neoplasms ,Disease ,Biology ,TRPC6 ,03 medical and health sciences ,0302 clinical medicine ,Mediator ,Breast cancer ,medicine ,TRPC6 Cation Channel ,Humans ,Molecular Biology ,Ion Transport ,Cell Biology ,medicine.disease ,Store-operated calcium entry ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,030220 oncology & carcinogenesis ,Identification (biology) ,Calcium ,Female ,Neuroscience ,Function (biology) - Abstract
TRPC6 channel is widely expressed in most human tissues and participates in a number of physiological processes. TRPC6 belongs to the DAG-activated subfamily of channels, but has also been postulated as a mediator in the store-operated calcium entry pathway. The recent characterization of TRPC6 crystal structure has granted a wonderful tool to finally dissect and understand TRPC6 physiological and biophysical properties. Growing evidences have demonstrated that the pattern of expression of TRPC6 proteins is upregulated in several pathophysiological conditions, including breast cancer. However, the real role of TRPC6 in breast cancer persists still unknown. Here we present the current state of the art concerning the function and significance of TRPC6 in this disease. Future investigations should be focus in the creation and identification of compounds that specifically target the channel to ameliorate TRPC6-related diseases.
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- 2020
9. PGRMC1 Inhibits Progesterone-Evoked Proliferation and Ca
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Carlos, Cantonero, Ginés M, Salido, Juan A, Rosado, and Pedro C, Redondo
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PGRMC1 ,ORAI1 Protein ,STIM2 ,Membrane Proteins ,Triple Negative Breast Neoplasms ,progesterone ,Article ,Ca2+-homeostasis ,Cell Line, Tumor ,Humans ,Calcium ,MDA-MB-231 cells ,Calcium Signaling ,Stromal Interaction Molecule 2 ,skin and connective tissue diseases ,Receptors, Progesterone ,Cell Proliferation ,TRPC Cation Channels - Abstract
Progesterone receptor membrane component 1 (PGRMC1) has been shown to regulate some cancer hallmarks. Progesterone (P4) evokes intracellular calcium (Ca2+) changes in the triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and BT-20) and in other breast cancer cell lines like the luminal MCF7 cells. PGRMC1 expression is elevated in MDA-MB-231 and MCF7 cells as compared to non-tumoral MCF10A cell line, and PGRMC1 silencing enhances P4-evoked Ca2+ mobilization. Here, we found a new P4-dependent Ca2+ mobilization pathway in MDA-MB-231 cells and other triple-negative breast cancer cells, as well as in MCF7 cells that involved Stromal interaction molecule 2 (STIM2), Calcium release-activated calcium channel protein 1 (Orai1), and Transient Receptor Potential Channel 1 (TRPC1). Stromal interaction molecule 1 (STIM1) was not involved in this novel Ca2+ pathway, as evidenced by using siRNA STIM1. PGRMC1 silencing reduced the negative effect of P4 on cell proliferation and cell death in MDA-MB-231 cells. In line with the latter observation, Nuclear Factor of Activated T-Cells 1 (NFAT1) nuclear accumulation due to P4 incubation for 48 h was enhanced in cells transfected with the small hairpin siRNA against PGRMC1 (shPGRMC1). These results provide evidence for a novel P4-evoked Ca2+ entry pathway that is downregulated by PGRMC1.
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- 2020
10. Non-coding RNAs and Ischemic Cardiovascular Diseases
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Tarik, Smani, Isabel, Mayoral-Gonzalez, Isabel, Galeano-Otero, Isabel, Gallardo-Castillo, Juan A, Rosado, Antonio, Ordoñez, and Abdelkrim, Hmadcha
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RNA, Untranslated ,Myocardium ,Myocardial Ischemia ,Humans ,Biomarkers - Abstract
The Ischemic Heart Disease (IHD) is considered a clinical condition characterized by myocardial ischemia causing an imbalance between myocardial blood supply and demand, leading to morbidity and mortality across the worldwide. Prompt diagnostic and prognostic represents key factors for the treatment and reduction of the mortality rate. Therefore, one of the newest frontiers in cardiovascular research is related to non-coding RNAs (ncRNAs), which prompted a huge interest in exploring ncRNAs candidates for utilization as potential therapeutic targets for diagnostic and prognostic and/or biomarkers in IHD. However, there are undoubtedly many more functional ncRNAs yet to be discovered and characterized. Here we will discuss our current knowledge and we will provide insight on the roles and effects elicited by some ncRNAs related to IHD.
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- 2020
11. Anticancer molecular mechanisms of oleocanthal
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Juan E. Quintero, Mohammed El Haouari, and Juan A. Rosado
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Antineoplastic Agents ,Cyclopentane Monoterpenes ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Phenols ,Neoplasms ,Oleocanthal ,medicine ,Humans ,STAT3 ,Olive Oil ,Pharmacology ,0303 health sciences ,Aldehydes ,biology ,Chemistry ,030302 biochemistry & molecular biology ,Cancer ,medicine.disease ,In vitro ,Apoptosis ,030220 oncology & carcinogenesis ,biology.protein ,STAT protein ,Cancer research ,Signal transduction ,Intracellular ,Signal Transduction - Abstract
Cancer is among the leading causes of mortality worldwide. Current cancer therapies are associated with serious side effects, which further damage patients' health. Therefore, the search for new anticancer agents with no toxic effects on normal and healthy cells is of great interest. Recently, we and other groups have demonstrated that oleocanthal (OLC), a phenolic compound from extra virgin olive oil, exhibits antitumor activity in various tumor models. However, the underlying mechanisms and intracellular targets of OLC remain to be completely elucidated. This review summarizes the current advancers concerning the anticancer activity of OLC, with particular emphasis on the molecular signaling pathways modulated by this compound in different tumor cell types. The major mechanisms of action of OLC include modulation of the apoptotic pathway, the HGF/c-Met pathway, and the signal transducer and activator of transcription 3 signaling pathway, among others. Furthermore, OLC has synergistic effects with anticancer drugs in vitro. Also discussed are OLC bioavailability and its concentration in olive oil. Data summarized here will represent a database for more extensive studies aimed at providing information on molecular mechanisms against cancer induced by OLC.
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- 2019
12. TRPC Channels: Dysregulation and Ca
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Débora, Falcón, Isabel, Galeano-Otero, Marta, Martín-Bórnez, María, Fernández-Velasco, Isabel, Gallardo-Castillo, Juan A, Rosado, Antonio, Ordóñez, and Tarik, Smani
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Transient Receptor Potential Channels ,cardiac infarction ,Ca2+ entry ,TRPC channel ,Myocardial Ischemia ,Animals ,Humans ,cardiac repair ,Calcium ,Myocytes, Cardiac ,Review ,Cardiovascular System ,Models, Biological - Abstract
Transient receptor potential canonical (TRPC) channels are ubiquitously expressed in excitable and non-excitable cardiac cells where they sense and respond to a wide variety of physical and chemical stimuli. As other TRP channels, TRPC channels may form homo or heterotetrameric ion channels, and they can associate with other membrane receptors and ion channels to regulate intracellular calcium concentration. Dysfunctions of TRPC channels are involved in many types of cardiovascular diseases. Significant increase in the expression of different TRPC isoforms was observed in different animal models of heart infarcts and in vitro experimental models of ischemia and reperfusion. TRPC channel-mediated increase of the intracellular Ca2+ concentration seems to be required for the activation of the signaling pathway that plays minor roles in the healthy heart, but they are more relevant for cardiac responses to ischemia, such as the activation of different factors of transcription and cardiac hypertrophy, fibrosis, and angiogenesis. In this review, we highlight the current knowledge regarding TRPC implication in different cellular processes related to ischemia and reperfusion and to heart infarction.
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- 2019
13. Filamin A Modulates Store-Operated Ca 2+ Entry by Regulating STIM1 (Stromal Interaction Molecule 1)–Orai1 Association in Human Platelets
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Jose Sanchez-Collado, Regis Bobe, Jose J. Lopez, Juan A. Rosado, Isaac Jardín, Letizia Albarran, Tarik Smani, Nuria Bermejo, and Pedro C. Redondo
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Blood Platelets ,inorganic chemicals ,0301 basic medicine ,Skin Neoplasms ,ORAI1 Protein ,Platelet Aggregation ,Filamins ,calcium signaling ,Filamin ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Serine ,Humans ,FLNA ,Protein Interaction Domains and Motifs ,Platelet ,Stromal Interaction Molecule 1 ,Melanoma ,Cytoskeleton ,Ion channel ,Calcium signaling ,phosphorylation ,ORAI1 ,Chemistry ,STIM1 ,Cyclic AMP-Dependent Protein Kinases ,Neoplasm Proteins ,Cell biology ,030104 developmental biology ,030220 oncology & carcinogenesis ,ion channel ,Phosphorylation ,Calcium ,Cardiology and Cardiovascular Medicine ,Ion Channel Gating ,Protein Binding - Abstract
Objective— Here, we provide evidence for the role of FLNA (filamin A) in the modulation of store-operated calcium entry (SOCE). Approach and Results— SOCE is a major mechanism for calcium influx controlled by the intracellular Ca 2+ stores. On store depletion, the endoplasmic reticulum calcium sensor STIM1 (stromal interaction molecule 1) redistributes into puncta at endoplasmic reticulum/plasma membrane junctions, a process supported by the cytoskeleton, where it interacts with the calcium channels; however, the mechanism for fine-tuning SOCE is not completely understood. Our results demonstrate that STIM1 interacts with FLNA on calcium store depletion in human platelets. The interaction is dependent on the phosphorylation of FLNA at Ser 2152 by the cAMP-dependent protein kinase. Impairment of FLNA phosphorylation and knockdown of FLNA expression using siRNA increased SOCE in platelets. Similarly, SOCE was significantly greater in FLNA-deficient melanoma M2 cells than in the FLNA-expressing M2 subclone A7. Expression of FLNA in M2 cells attenuated SOCE, an effect prevented when the cells were transfected with the nonphosphorylatable FLNA S2152A mutant. Transfection of M2 cells with the STIM1(K684,685E) mutant reduced the STIM1–FLNA interaction. In platelets, attenuation of FLNA expression using siRNA resulted in enhanced association of STIM1 with the cytoskeleton, greater STIM1–Orai1 interaction, and SOCE. Introduction of an anti-FLNA (2597–2647) antibody attenuated the STIM1–FLNA interaction and enhanced thrombin-induced platelet aggregation. Conclusions— Our results indicate that FLNA modulates SOCE and then the correct platelet function, by fine-tuning the distribution of STIM1 in the cytoskeleton and the interaction with Orai1 channels.
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- 2018
14. Role of Orai3 in the Pathophysiology of Cancer
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Ginés M. Salido, Victor Ronco, Jose Sanchez-Collado, Jose J. Lopez, Natalia Prevarskaya, Isaac Jardin, Charlotte Dubois, and Juan A. Rosado
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QH301-705.5 ,Colorectal cancer ,Apoptosis ,calcium entry ,Review ,Catalysis ,Inorganic Chemistry ,Prostate cancer ,chemistry.chemical_compound ,Cell Movement ,Neoplasms ,medicine ,Animals ,Humans ,cancer ,Calcium Signaling ,Biology (General) ,Physical and Theoretical Chemistry ,QD1-999 ,Molecular Biology ,Spectroscopy ,Cell Proliferation ,orai3 ,orai1 ,Leukotriene C4 ,ORAI1 ,Cell Cycle ,Organic Chemistry ,Cancer ,STIM1 ,General Medicine ,STIM2 ,medicine.disease ,Computer Science Applications ,Chemistry ,chemistry ,Cancer research ,Adenocarcinoma ,Calcium ,Calcium Channels - Abstract
The mammalian exclusive Orai3 channel participates in the generation and/or modulation of two independent Ca2+ currents, the store-operated current, Icrac, involving functional interactions between the stromal interaction molecules (STIM), STIM1/STIM2, and Orai1/Orai2/Orai3, as well as the store-independent arachidonic acid (AA) (or leukotriene C4)-regulated current Iarc, which involves Orai1, Orai3 and STIM1. Overexpression of functional Orai3 has been described in different neoplastic cells and cancer tissue samples as compared to non-tumor cells or normal adjacent tissue. In these cells, Orai3 exhibits a cell-specific relevance in Ca2+ influx. In estrogen receptor-positive breast cancer cells and non-small cell lung cancer (NSCLC) cells store-operated Ca2+ entry (SOCE) is strongly dependent on Orai3 expression while in colorectal cancer and pancreatic adenocarcinoma cells Orai3 predominantly modulates SOCE. On the other hand, in prostate cancer cells Orai3 expression has been associated with the formation of Orai1/Orai3 heteromeric channels regulated by AA and reduction in SOCE, thus leading to enhanced proliferation. Orai3 overexpression is associated with supporting several cancer hallmarks, including cell cycle progression, proliferation, migration, and apoptosis resistance. This review summarizes the current knowledge concerning the functional role of Orai3 in the pathogenesis of cancer.
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- 2021
15. TMEM97 facilitates the activation of SOCE by downregulating the association of cholesterol to Orai1 in MDA-MB-231 cells
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Carlos Cantonero, Ginés M. Salido, Pedro J. Camello, Pedro C. Redondo, and Juan A. Rosado
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0301 basic medicine ,ORAI1 Protein ,Down-Regulation ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,Humans ,Gene silencing ,skin and connective tissue diseases ,Molecular Biology ,Regulation of gene expression ,Chemistry ,Cholesterol ,ORAI1 ,Calcium channel ,Membrane Proteins ,STIM1 ,Cell Biology ,Transfection ,Cell biology ,Protein Transport ,030104 developmental biology ,Cell culture ,030220 oncology & carcinogenesis ,Calcium - Abstract
The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced in some tumour cells. We have recently shown that TMEM97 is involved in the proliferation of the breast cancer cell line MDA-MB-231, probably through changes in store-operated calcium entry (SOCE). By using silencing and overexpression of TMEM97 in MDA-MB-231 cells (two manoeuvres that either reduce or increase the calcium influx, respectively), we show enhanced cholesterol uptake in these cells as compared to the non-tumoral breast cell line, MCF10A. The enhanced cholesterol uptake in MDA-MB-231 cells was inhibited by silencing TMEM97, while overexpression of this protein increased cholesterol uptake in MCF10A cells and, therefore, indicating that this protein plays a role in the enhanced cholesterol uptake in MDA-MB-231 cancer cell line. TMEM97 silencing and overexpression resulted in an increase and decrease in the association of cholesterol to the SOCE calcium channel Orai1, respectively. Interestingly, silencing of TMEM97 in MDA-MB-231 cells significantly reduced the co-localization of Orai1 with the SOCE regulatory protein STIM1. Finally, neither silencing nor overexpression of TMEM97 altered SOCE in MDA-MB-231 cells transfected with the cholesterol insensible mutant of Orai1(Y80E). Our results reveal a novel regulatory mechanism of SOCE that relies on TMEM97 activity that courses through the reduction of the cholesterol content in the plasma membrane, and subsequently, by impairing its interaction with Orai1.
- Published
- 2021
16. The Orai1-AC8 Interplay: How Breast Cancer Cells Escape from Orai1 Channel Inactivation
- Author
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Jose J. Lopez, Juan A. Rosado, and Jose Sanchez-Collado
- Subjects
Orai1 ,ORAI1 Protein ,QH301-705.5 ,Cell ,Triple Negative Breast Neoplasms ,AC8 ,Review ,Adenylyl cyclase ,chemistry.chemical_compound ,medicine ,Humans ,inactivation ,Biology (General) ,triple-negative breast cancer cells ,Chemistry ,Mechanism (biology) ,ORAI1 ,Cancer ,General Medicine ,medicine.disease ,Cell biology ,medicine.anatomical_structure ,Pka signaling ,Phosphorylation ,Calcium ,Female ,Calcium Channels ,Breast cancer cells ,Adenylyl Cyclases ,SOCE - Abstract
The interplay between the Ca2+-sensitive adenylyl cyclase 8 (AC8) and Orai1 channels plays an important role both in the activation of the cAMP/PKA signaling and the modulation of Orai1-dependent Ca2+ signals. AC8 interacts with a N-terminal region that is exclusive to the Orai1 long variant, Orai1α. The interaction between both proteins allows the Ca2+ that enters the cell through Orai1α to activate the generation of cAMP by AC8. Subsequent PKA activation results in Orai1α inactivation by phosphorylation at serine-34, thus shaping Orai1-mediated cellular functions. In breast cancer cells, AC8 plays a relevant role supporting a variety of cancer hallmarks, including proliferation and migration. Breast cancer cells overexpress AC8, which shifts the AC8-Orai1 stoichiometry in favor of the former and leads to the impairment of PKA-dependent Orai1α inactivation. This mechanism contributes to the enhanced SOCE observed in triple-negative breast cancer cells. This review summarizes the functional interaction between AC8 and Orai1α in normal and breast cancer cells and its relevance for different cancer features.
- Published
- 2021
17. Involvement of stanniocalcins in the deregulation of glycaemia in obese mice and type 2 diabetic patients
- Author
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Carlos Cantonero Chamorro, María Jose Hernández, Pedro C. Redondo, Luis Gómez-Gordo, María P. Granados, Francisca Jiménez Jiménez, Antonio Jesús Corbacho, María José Tarancón Rubio, Rocío González Montero, María José Cazorla González, Manuel Pérez Martínez, Jose J. Lopez, Isaac Jardin, José María Brull, Elena Delgado, Manuel Luis Duran, Maria Reyes Panadero, and Juan A. Rosado
- Subjects
Adult ,Blood Glucose ,0301 basic medicine ,medicine.medical_specialty ,DM2 ,medicine.medical_treatment ,Mice, Obese ,STC1 and STC2 ,Glucagon ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Diabetes mellitus ,medicine ,Animals ,Humans ,STC1 ,Pancreas ,Aged ,Glycoproteins ,geography ,geography.geographical_feature_category ,business.industry ,Pancreatic islets ,Insulin ,Intracellular Signaling Peptides and Proteins ,Organ Size ,Original Articles ,Cell Biology ,Middle Aged ,Islet ,medicine.disease ,Mice, Inbred C57BL ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,Diabetes Mellitus, Type 2 ,030220 oncology & carcinogenesis ,deregulated glycaemia ,Intercellular Signaling Peptides and Proteins ,Molecular Medicine ,Original Article ,business ,Immunostaining - Abstract
Stanniocalcins are expressed in the pancreas tissue, and it was suggested a direct correlation between circulating insulin and STC2 concentrations in human. Here, we show a significant correlation between STC1 and both glycaemia and glycosylated haemoglobin among DM2 patients, while DM2 patients who present the greatest glycosylated haemoglobin values exhibited the lowest STC2 expression. However, treatment of patients with antiglycaemic drugs does not significantly modify the expression of both STCs. On the other hand, STC2‐/‐ mice that exhibited neonatal and adult overweight further presented deregulated glycaemia when they were feed with a hypercaloric diet (breeding pellet, BP). This alteration is more evident at the early stages of the animal life. Deregulated glycaemia in these mice was confirmed using glucose oral test. In addition, STC2‐/‐ mice present enhanced pancreas size; thus, the histological analysis reveals that WT mice respond to BP diet by increasing the size of the pancreatic islets through inducing cell division, and STC2‐/‐ mice lack this compensatory mechanism. Contrary, BP fed STC2‐/‐ mice show enhanced number of islets but of similar size than those fed with regular pellet. Histopathological analysis demonstrates tissue structure disruption and erythrocytes infiltrations in STC2‐/‐ mice, possibly due to the stress evoked by the BP diet. Finally, enhanced glucagon immunostaining was observed in the islet of STC2‐/‐ mice, and the glucagon ELISA assay confirmed the increase in the circulating glucagon. Summarizing, we present evidence of the role of STCs, mainly STC2, as a possible early marker during development of diabetes mellitus.
- Published
- 2017
18. Orai1 and Orai2 mediate store-operated calcium entry that regulates HL60 cell migration and FAK phosphorylation
- Author
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Juan A. Rosado, Raquel Diez-Bello, Ginés M. Salido, and Isaac Jardin
- Subjects
0301 basic medicine ,ORAI1 Protein ,ORAI2 Protein ,HL-60 Cells ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,TRPC3 ,Humans ,Gene silencing ,Phosphorylation ,Molecular Biology ,Cell Proliferation ,Gene knockdown ,Ion Transport ,Cell growth ,Tyrosine phosphorylation ,Cell Biology ,Store-operated calcium entry ,Cell biology ,030104 developmental biology ,chemistry ,Focal Adhesion Protein-Tyrosine Kinases ,Calcium ,Intracellular - Abstract
Store-operated Ca2+ entry (SOCE) is a major mechanism for the regulation of intracellular Ca2+ homeostasis and cellular function. Emerging evidence has revealed that altered expression and function of the molecular determinants of SOCE play a critical role in the development or maintenance of several cancer hallmarks, including enhanced proliferation and migration. Here we show that, in the acute myeloid leukemia cell line HL60, Orai2 is highly expressed at the transcript level, followed by the expression of Orai1. Using fluorescence Ca2+ imaging we found that Orai2 silencing significantly attenuated thapsigargin-induced SOCE, as well as knockdown of Orai1, while silencing the expression of both channels almost completely reduced SOCE, thus suggesting that SOCE in these cells is strongly dependent on Orai1 and Orai2. On the other hand, the expression of TRPC1, TRPC3 and TRPC6 is almost absent at the transcript and protein level. Bromodeoxyuridine cell proliferation assay revealed that Orai1 and Orai2 expression silencing significantly reduced HL60 cell proliferation. Furthermore, knockdown of Orai1 and Orai2 significantly attenuated the ability of HL60 to migrate in vitro as determined by transwell migration assay, probably due to the impairment of FAK tyrosine phosphorylation. These findings provide evidence for a role for Orai1 and Orai2, in SOCE and migration in the human HL60 promyeloblastic cell line. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
- Published
- 2017
19. Pathophysiological Significance of Store-Operated Calcium Entry in Cardiovascular and Skeletal Muscle Disorders and Angiogenesis
- Author
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Javier, Avila-Medina, Isabel, Mayoral-González, Isabel, Galeano-Otero, Pedro C, Redondo, Juan A, Rosado, and Tarik, Smani
- Subjects
Ion Transport ,Muscular Diseases ,Neovascularization, Pathologic ,Cardiovascular Diseases ,Humans ,Calcium ,Calcium Channels ,Calcium Signaling - Abstract
Store-Operated Ca
- Published
- 2019
20. Special Issue on New Cellular, Genetic and Proteomic Tools in the Prevention and Management of Diabetes Mellitus
- Author
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Pedro Cosme Redondo Liberal and Juan A. Rosado
- Subjects
Pharmacology ,Proteomics ,business.industry ,Organic Chemistry ,Proteins ,Computational biology ,medicine.disease ,Biochemistry ,Diabetes Mellitus, Type 1 ,Diabetes Mellitus, Type 2 ,Diabetes mellitus ,Drug Discovery ,medicine ,Molecular Medicine ,Humans ,business - Published
- 2019
21. Inactivation of Proprotein Convertases in T Cells Inhibits PD-1 Expression and Creates a Favorable Immune Microenvironment in Colorectal Cancer
- Author
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Frédéric Delom, Abdel-Majid Khatib, Jone Olaizola, Jose J. Lopez, Marielle Dubreuil, Angela Pappalardo, Amandine Mouchard, Julie Déchanet-Merville, Mariane Fonck, François Ghiringhelli, Yannick Leger, Juan A. Rosado, Serge Evrard, Fabienne Soulet, Geraldine Siegfried, Mercedes Tomé, Delphine Fessart, Isabelle Soubeyran, Vincent Pitard, Dominique Béchade, Laboratoire Angiogenèse et Micro-environnement des Cancers (LAMC), Université Sciences et Technologies - Bordeaux 1-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunology from Concept and Experiments to Translation (ImmunoConcept), Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB), Composantes innées de la réponse immunitaire et différenciation (CIRID), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Institut National de l'Environnement Industriel et des Risques (INERIS), Centre de recherche Cardio-Thoracique de Bordeaux [Bordeaux] (CRCTB), Université Bordeaux Segalen - Bordeaux 2-CHU Bordeaux [Bordeaux]-Institut National de la Santé et de la Recherche Médicale (INSERM), Département d'oncologie digestive, Institut Bergonié [Bordeaux], UNICANCER-UNICANCER, Lipides - Nutrition - Cancer (U866) (LNC), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA), Génétique tumorale - Pathologie, CHU Bordeaux [Bordeaux]-Institut Bergonié [Bordeaux], Hémostase et thrombose, Institut National de la Santé et de la Recherche Médicale (INSERM), Head of Digestive Tumours Unit and Department of Surgery, Université Bordeaux Segalen - Bordeaux 2, Laboratoire de pharmacologie expérimentale et clinique : cibles moléculaires en cancérologie ((U 716)), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)
- Subjects
0301 basic medicine ,MAPK/ERK pathway ,Cancer Research ,Colorectal cancer ,medicine.medical_treatment ,Programmed Cell Death 1 Receptor ,Mice, Nude ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Mice ,03 medical and health sciences ,Lymphocytes, Tumor-Infiltrating ,0302 clinical medicine ,Tumor Microenvironment ,medicine ,Animals ,Humans ,Cytotoxic T cell ,Furin ,ComputingMilieux_MISCELLANEOUS ,Mice, Inbred BALB C ,biology ,Chemistry ,NFAT ,Immunotherapy ,medicine.disease ,3. Good health ,CTL ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,Heterografts ,[SDV.IMM]Life Sciences [q-bio]/Immunology ,Proprotein Convertases ,Colorectal Neoplasms ,T-Lymphocytes, Cytotoxic - Abstract
Proprotein convertases (PC) activate precursor proteins that play crucial roles in various cancers. In this study, we investigated whether PC enzyme activity is required for expression of the checkpoint protein programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTL) in colon cancer. Although altered expression of the PC secretory pathway was observed in human colon cancers, only furin showed highly diffuse expression throughout the tumors. Inhibition of PCs in T cells using the general protein-based inhibitor α1-PDX or the pharmacologic inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethylketone repressed PD-1 and exhausted CTLs via induction of T-cell proliferation and apoptosis inhibition, which improved CTL efficacy against microsatellite instable and microsatellite stable colon cancer cells. In vivo, inhibition of PCs enhanced CTL infiltration in colorectal tumors and increased tumor clearance in syngeneic mice compared with immunodeficient mice. Inhibition of PCs repressed PD-1 expression by blocking proteolytic maturation of the Notch precursor, inhibiting calcium/NFAT and NF-κB signaling, and enhancing ERK activation. These findings define a key role for PCs in regulating PD-1 expression and suggest targeting PCs as an adjunct approach to colorectal tumor immunotherapy. Significance: Protein convertase enzymatic activity is required for PD-1 expression on T cells, and inhibition of protein convertase improves T-cell targeting of microsatellite instable and stable colorectal cancer.
- Published
- 2019
22. Virulence properties of three new Photobacterium species affecting cultured fish
- Author
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Miguel Balado, Alejandro M. Labella, Juan J. Rosado, Juan J. Borrego, and Manuel L. Lemos
- Subjects
Lipopolysaccharide ,Virulence Factors ,Virulence ,Aquaculture ,Applied Microbiology and Biotechnology ,Microbiology ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Fish Diseases ,Vibrionaceae ,Extracellular ,Animals ,Humans ,Thermolabile ,Cytotoxicity ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,Photobacterium ,General Medicine ,biology.organism_classification ,Sea Bream ,chemistry ,Cell culture ,Gram-Negative Bacterial Infections ,Biotechnology - Abstract
Aims: Several virulence factors of three new Photobacterium species: Photobacterium toruni, Photobacterium malacitanum and Photobacterium andalusiense associated with diseases of cultured redbanded seabream (Pagrus auriga) were studied. The exoenzymatic activities, adherence and cytotoxic capabilities, and iron-uptake mechanisms were determined both in bacterial extracellular products (ECP) and whole bacterial cells. The histopathology damages provoked on redbanded seabream by the ECP was also studied. Methods and Results: The highest exoenzymatic activities of the ECP were alkaline- and acid-phosphatase, phosphohydrolase and lipase. The ECP were strongly lethal for fish at 4–96 h post-inoculation (p.i). Histological changes were evident at 96 hpi of ECP, affecting head kidney, splenic parenchyma and heart. Cytotoxicity assays, on three fish lines and one human cell line, were conducted using whole bacterial cells and their ECP. The new species tested were cytotoxic only for fish cell lines using whole bacterial cells. Bacterial adherence showed an adherence index moderate on CHSE-214 cell line. All strains showed variable haemolytic activity, and were able to grow under iron-limiting conditions, although the CAS reactivitiy was very low. However, all strains produced high amounts of extracelullar citrate that could be used as iron carrier, and use haem as iron source, except the P. toruni strains because a deletion in the genomic region encoding this ability in all Vibrionaceae members. Conclusions: The toxic activity of the bacterial ECPs was thermolabile, and not associated with their thermoresistant lipopolysaccharide content. The virulence of the strains tested could not be related to the haemolytic activity. Iron uptake could be based on the use of endogenous citrate as iron carrier and P. toruni lacks the ability to use haem as iron source. Significance and Impact of the study: The study analyses for the first time the virulence properties of three new species of Photobacterium pathogenic for fish.
- Published
- 2019
23. STIM1 phosphorylation at Y316 modulates its interaction with SARAF and the activation of SOCE and ICRAC
- Author
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Juan A. Rosado, Irene Frischauf, Esther Lopez, Ginés M. Salido, Tarik Smani, Carlos Cantonero, Isaac Jardin, Martin Muik, Pedro C. Redondo, and Isabella Derler
- Subjects
inorganic chemicals ,Orai1 ,ORAI1 Protein ,STIM1 ,Biology ,Tyrosine phosphorylation ,03 medical and health sciences ,chemistry.chemical_compound ,Intracellular Calcium-Sensing Proteins ,0302 clinical medicine ,Y316 ,Animals ,Humans ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Phosphorylation ,SARAF ,030304 developmental biology ,0303 health sciences ,Gene knockdown ,ICRAC ,ORAI1 ,HEK 293 cells ,Membrane Proteins ,Colocalization ,Cell Biology ,Transfection ,Calcium Release Activated Calcium Channels ,Neoplasm Proteins ,Cell biology ,HEK293 Cells ,chemistry ,Tyrosine ,Calcium ,030217 neurology & neurosurgery - Abstract
Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store-operated Ca2+ entry (SOCE). Hence, identification of the relevant phosphorylatable STIM1 residues with a possible role in the regulation of STIM1 function and SOCE is of interest. By performing a computational analysis, we identified that the Y316 residue is susceptible to phosphorylation. Expression of the STIM1-Y316F mutant in HEK293, NG115-401L and MEG-01 cells resulted in a reduction in STIM1 tyrosine phosphorylation, SOCE and the Ca2+ release-activated Ca2+ current (ICRAC). STIM1-Orai1 colocalization was reduced in HEK293 cells transfected with YFP-STIM1-Y316F compared to in cells with wild-type (WT) YFP-tagged STIM1. Additionally, the Y316F mutation altered the pattern of interaction between STIM1 and SARAF under resting conditions and upon Ca2+ store depletion. Expression of the STIM1 Y316F mutant enhanced slow Ca2+-dependent inactivation (SCDI) as compared to STIM1 WT, an effect that was abolished by SARAF knockdown. Finally, in NG115-401L cells transfected with shRNA targeting SARAF, expression of STIM1 Y316F induced greater SOCE than STIM1 WT. Taken together, our results provide evidence supporting the idea that phosphorylation of STIM1 at Y316 plays a relevant functional role in the activation and modulation of SOCE.
- Published
- 2019
24. EFHB is a Novel Cytosolic Ca2+ Sensor That Modulates STIM1-SARAF Interaction
- Author
-
Tarik Smani, Jose J. Lopez, Alejandro Berna-Erro, Ginés M. Salido, Pedro J. Camello, Letizia Albarran, Jose Sanchez-Collado, Isaac Jardin, Juan A. Rosado, and Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica
- Subjects
0301 basic medicine ,inorganic chemicals ,Orai1 ,Physiology ,STIM1 ,lcsh:Physiology ,Calcium entry ,lcsh:Biochemistry ,03 medical and health sciences ,Cytosol ,Intracellular Calcium-Sensing Proteins ,EFHB ,Humans ,lcsh:QD415-436 ,Protein Interaction Maps ,Stromal Interaction Molecule 1 ,EF Hand Motifs ,SARAF ,Physics ,lcsh:QP1-981 ,Membrane Proteins ,Neoplasm Proteins ,030104 developmental biology ,HEK293 Cells ,Calcium ,Humanities ,HeLa Cells - Abstract
Background/aims: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Among the proteins involved in the regulation of SOCE, SARAF prevents spontaneous activation of SOCE and modulates STIM1 function. Methods: Cytosolic Ca2+ mobilization was estimated in fura-2-loaded cells using an epifluorescence inverted microscope. STIM1 interaction with Orai1, EFHB (EF-hand domain family member B, also known as CFAP21) and SARAF was detected by immunoprecipitation followed by Western blotting using specific antibodies. The involvement of EFHB in the translocation of NFAT to the nucleus was detected by confocal microscopy. Results: Here, we report the identification of EFHB as a new SOCE regulator. EFHB interacts with STIM1 upon store depletion and dissociates through a Ca2+-dependent mechanism. RNAi-mediated silencing as well as overexpression studies revealed that EFHB plays a relevant role in the interaction of STIM1 and Orai1 upon store depletion, the activation of SOCE and NFAT translocation from the cytosol to the nucleus. Silencing EFHB expression abolished the dissociation of SARAF from STIM1, which indicates that EFHB might play an important role in the dynamic interaction between both proteins, which is relevant for the activation of Orai1 channels upon Ca2+ store depletion and their subsequent modulation via slow Ca2+-dependent inactivation. Conclusion: Our results indicate that EFHB is a new SOCE regulator that modulates STIM1-SARAF interaction. MINECO BFU2013-45564-C2-1-P/2-P, BFU2016-74932-C2-1-P/2-P, BFU2016-74932-C2-1-P Junta de Extremadura-FEDER (Fondo Europeo de Desarrollo Regional Grants) IB16046, GR18061 Junta de Extremadura-FEDER European Union (EU) IB16046 Ministerio Economía y Competitividad, España IJCI-2015-25665 MINECO Grant BFU2016-74932-C2-1-P
- Published
- 2018
25. Store-Operated Ca
- Author
-
Isaac, Jardin, Jose J, Lopez, Gines M, Salido, and Juan A, Rosado
- Subjects
Orai1 ,Epithelial-Mesenchymal Transition ,STIM1 ,MDA-MB-231 ,proliferation ,MCF7 ,Breast Neoplasms ,calcium entry ,Review ,migration ,TRPC channels ,breast cancer ,Cell Movement ,Humans ,Female ,Neoplasm Invasiveness ,Calcium Channels ,Calcium Signaling ,Cell Proliferation - Abstract
Breast cancer is the most common type of cancer in women. It is a heterogeneous disease that ranges from the less undifferentiated luminal A to the more aggressive basal or triple negative breast cancer molecular subtype. Ca2+ influx from the extracellular medium, but more specifically store-operated Ca2+ entry (SOCE), has been reported to play an important role in tumorigenesis and the maintenance of a variety of cancer hallmarks, including cell migration, proliferation, invasion or epithelial to mesenchymal transition. Breast cancer cells remodel the expression and functional role of the molecular components of SOCE. This review focuses on the functional role and remodeling of SOCE in breast cancer cells. The current studies suggest the need to deepen our understanding of SOCE in the biology of the different breast cancer subtypes in order to develop new and specific therapeutic strategies.
- Published
- 2018
26. Molecular modulators of store-operated calcium entry
- Author
-
Ginés M. Salido, Tarik Smani, Luis Gómez, Letizia Albarran, Juan A. Rosado, and Jose J. Lopez
- Subjects
inorganic chemicals ,0301 basic medicine ,Orai1 ,ORAI1 Protein ,STIM1 ,Protein Conformation ,Endoplasmic Reticulum ,Models, Biological ,03 medical and health sciences ,Intracellular Calcium-Sensing Proteins ,Animals ,Humans ,Septin ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Molecular Biology ,SARAF ,TRPC ,TRPC Cation Channels ,SOC channels ,ORAI1 ,Chemistry ,Endoplasmic reticulum ,ORMDL3 ,Membrane Proteins ,Store-operated calcium entry ,Cell Biology ,Calcium Release Activated Calcium Channels ,Cell biology ,Protein Subunits ,Electrophysiology ,Golli ,030104 developmental biology ,CRACR2A ,Calcium ,Calcium Channels ,Intracellular - Abstract
Three decades ago, store-operated Ca 2 + entry (SOCE) was identified as a unique mechanism for Ca 2 + entry through plasma membrane (PM) Ca 2 + -permeable channels modulated by the intracellular Ca 2 + stores, mainly the endoplasmic reticulum (ER). Extensive analysis of the communication between the ER and the PM leads to the identification of the protein STIM1 as the ER-Ca 2 + sensor that gates the Ca 2 + channels in the PM. Further analysis on the biophysical, electrophysiological and biochemical properties of STIM1-dependent Ca 2 + channels has revealed the presence of a highly Ca 2 + -selective channel termed Ca 2 + release-activated Ca 2 + channel (CRAC), consisting of Orai1 subunits, and non-selective cation channels named store-operated channels (SOC), including both Orai1 and TRPC channel subunits. Since the identification of the key elements of CRAC and SOC channels a number of intracellular modulators have been reported to play essential roles in the stabilization of STIM–Orai interactions, collaboration with STIM1 conformational changes or mediating slow Ca 2 + -dependent inactivation. Here, we review our current understanding of some of the key modulators of STIM1–Orai1 interaction, including the proteins CRACR2A, STIMATE, SARAF, septins, golli and ORMDL3.
- Published
- 2016
27. Store-operated Ca2+ Entry-associated Regulatory factor (SARAF) Plays an Important Role in the Regulation of Arachidonate-regulated Ca2+ (ARC) Channels
- Author
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Jose J. Lopez, Ginés M. Salido, Juan A. Rosado, Geoffrey E. Woodard, and Letizia Albarran
- Subjects
0301 basic medicine ,ORAI1 Protein ,Cell Survival ,Biology ,Endoplasmic Reticulum ,Biochemistry ,Cell membrane ,03 medical and health sciences ,Intracellular Calcium-Sensing Proteins ,0302 clinical medicine ,Cell Line, Tumor ,medicine ,Humans ,Stromal Interaction Molecule 1 ,RNA, Small Interfering ,Molecular Biology ,Ion transporter ,Neurons ,Arachidonic Acid ,Ion Transport ,Arc (protein) ,Voltage-dependent calcium channel ,ORAI1 ,Endoplasmic reticulum ,Cell Membrane ,Membrane Proteins ,STIM1 ,Cell Biology ,Neoplasm Proteins ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Calcium ,Calcium Channels ,Signal transduction ,030217 neurology & neurosurgery ,Signal Transduction ,Protein Binding - Abstract
The store-operated Ca(2+)entry-associated regulatory factor (SARAF) has recently been identified as a STIM1 regulatory protein that facilitates slow Ca(2+)-dependent inactivation of store-operated Ca(2+)entry (SOCE). Both the store-operated channels and the store-independent arachidonate-regulated Ca(2+)(ARC) channels are regulated by STIM1. In the present study, we show that, in addition to its location in the endoplasmic reticulum, SARAF is constitutively expressed in the plasma membrane, where it can interact with plasma membrane (PM)-resident ARC forming subunits in the neuroblastoma cell line SH-SY5Y. Using siRNA-based and overexpression approaches we report that SARAF negatively regulates store-independent Ca(2+)entry via the ARC channels. Arachidonic acid (AA) increases the association of PM-resident SARAF with Orai1. Finally, our results indicate that SARAF modulates the ability of AA to promote cell survival in neuroblastoma cells. In addition to revealing new insight into the biology of ARC channels in neuroblastoma cells, these findings provide evidence for an unprecedented location of SARAF in the plasma membrane.
- Published
- 2016
28. Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry
- Author
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Ginés M. Salido, Carlos Cantonero, Juan A. Rosado, Pedro C. Redondo, Jose J. Lopez, and Jose Sanchez-Collado
- Subjects
Programmed cell death ,ORAI1 Protein ,Orai3 ,Cell Survival ,Antineoplastic Agents ,Breast Neoplasms ,Article ,Catalysis ,lcsh:Chemistry ,Inorganic Chemistry ,Phospholipase A2 ,Cell Movement ,Cell Line, Tumor ,Humans ,MDA-MB-231 cells ,Stromal Interaction Molecule 1 ,Physical and Theoretical Chemistry ,lcsh:QH301-705.5 ,Molecular Biology ,ARC channels ,Spectroscopy ,Cell Proliferation ,Arachidonic Acid ,Arc (protein) ,biology ,Cell growth ,ORAI1 ,Chemistry ,Organic Chemistry ,Depolarization ,General Medicine ,Neoplasm Proteins ,Computer Science Applications ,Cell biology ,Gene Expression Regulation, Neoplastic ,lcsh:Biology (General) ,lcsh:QD1-999 ,Apoptosis ,MCF-7 Cells ,biology.protein ,Calcium ,Female ,Calcium Channels ,Intracellular - Abstract
Arachidonic acid (AA) is a phospholipase A2 metabolite that has been reported to mediate a plethora of cellular mechanisms involved in healthy and pathological states such as platelet aggregation, lymphocyte activation, and tissue inflammation. AA has been described to activate Ca2+ entry through the arachidonate-regulated Ca2+-selective channels (ARC channels). Here, the analysis of the changes in the intracellular Ca2+ homeostasis revealed that, despite MDA-MB-231 cells expressing the ARC channel components Orai1, Orai3, and STIM1, AA does not evoke Ca2+ entry in these cells. We observed that AA evokes Ca2+ entry in MDA-MB-231 cells transiently expressing ARC channels. Nevertheless, MDA-MB-231 cell treatment with AA reduces cell proliferation and migration while inducing cell death through apoptosis. The latter mostly likely occurs via mitochondria membrane depolarization and the activation of caspases-3, -8, and -9. Altogether, our results indicate that AA exerts anti-tumoral effects on MDA-MB-231 cells, without having any effect on non-tumoral breast epithelial cells, by a mechanism that is independent on the activation of Ca2+ influx via ARC channels.
- Published
- 2020
29. TRPC Channels: Dysregulation and Ca2+ Mishandling in Ischemic Heart Disease
- Author
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Isabel Galeano-Otero, Tarik Smani, Isabel Gallardo-Castillo, Antonio Ordóñez, Juan A. Rosado, María Fernández-Velasco, Marta Martín-Bórnez, Débora Falcón, and Universidad de Sevilla. Departamento de Estomatología
- Subjects
0301 basic medicine ,Gene isoform ,Myocardial Ischemia ,Ischemia ,030204 cardiovascular system & hematology ,Cardiovascular System ,Models, Biological ,Calcium in biology ,Cardiac repair ,03 medical and health sciences ,Transient receptor potential channel ,Transient Receptor Potential Channels ,0302 clinical medicine ,Cardiac infarction ,medicine ,Animals ,Humans ,Myocytes, Cardiac ,ca2+ entry ,lcsh:QH301-705.5 ,TRPC ,Ion channel ,Chemistry ,General Medicine ,medicine.disease ,trpc channel ,Cell biology ,030104 developmental biology ,cardiac infarction ,lcsh:Biology (General) ,Ca2+ entry ,physiology ,TRPC channel ,cardiac repair ,Calcium ,Signal transduction ,Intracellular - Abstract
Transient receptor potential canonical (TRPC) channels are ubiquitously expressed in excitable and non-excitable cardiac cells where they sense and respond to a wide variety of physical and chemical stimuli. As other TRP channels, TRPC channels may form homo or heterotetrameric ion channels, and they can associate with other membrane receptors and ion channels to regulate intracellular calcium concentration. Dysfunctions of TRPC channels are involved in many types of cardiovascular diseases. Significant increase in the expression of different TRPC isoforms was observed in different animal models of heart infarcts and in vitro experimental models of ischemia and reperfusion. TRPC channel-mediated increase of the intracellular Ca2+ concentration seems to be required for the activation of the signaling pathway that plays minor roles in the healthy heart, but they are more relevant for cardiac responses to ischemia, such as the activation of different factors of transcription and cardiac hypertrophy, fibrosis, and angiogenesis. In this review, we highlight the current knowledge regarding TRPC implication in different cellular processes related to ischemia and reperfusion and to heart infarction.
- Published
- 2020
30. TRPC Channels in the SOCE Scenario
- Author
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Ginés M. Salido, Jose J. Lopez, Juan A. Rosado, Isaac Jardin, Tarik Smani, Jose Sanchez-Collado, and Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica
- Subjects
inorganic chemicals ,Functional role ,Orai1 ,Cell type ,ORAI1 Protein ,TRPC1 ,STIM1 ,Review ,Models, Biological ,Biophysical Phenomena ,Transient receptor potential channel ,store-operated Ca2+ entry (SOCE) ,Calcium influx ,Animals ,Humans ,Stromal Interaction Molecule 1 ,stim1 ,lcsh:QH301-705.5 ,TRPC ,TRPC Cation Channels ,orai1 ,ORAI1 ,Chemistry ,store-operated ca2+ entry (soce) ,General Medicine ,Cell biology ,trpc1 ,lcsh:Biology (General) ,Calcium Channels ,Store-operated Ca2+ entry (SOCE) ,calcium influx ,Function (biology) - Abstract
Transient receptor potential (TRP) proteins form non-selective Ca2+ permeable channels that contribute to the modulation of a number of physiological functions in a variety of cell types. Since the identification of TRP proteins in Drosophila, it is well known that these channels are activated by stimuli that induce PIP2 hydrolysis. The canonical TRP (TRPC) channels have long been suggested to be constituents of the store-operated Ca2+ (SOC) channels; however, none of the TRPC channels generate Ca2+ currents that resemble ICRAC. STIM1 and Orai1 have been identified as the components of the Ca2+ release-activated Ca2+ (CRAC) channels and there is a body of evidence supporting that STIM1 is able to gate Orai1 and TRPC1 in order to mediate non-selective cation currents named ISOC. STIM1 has been found to interact to and activate Orai1 and TRPC1 by different mechanisms and the involvement of TRPC1 in store-operated Ca2+ entry requires both STIM1 and Orai1. In addition to the participation of TRPC1 in the ISOC currents, TRPC1 and other TRPC proteins might play a relevant role modulating Orai1 channel function. This review summarizes the functional role of TRPC channels in the STIM1–Orai1 scenario. Junta de Extremadura Consejería de Economía e Infraestructura-FEDER Grant IB16046 y GR18061 Junta de Extremadura TA18011 y TA18054 Ministerio de Ciencia, Innovación y Universidades
- Published
- 2020
31. Filamin A modulates platelet function
- Author
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Juan A. Rosado, Isaac Jardin, and Jose J. Lopez
- Subjects
0301 basic medicine ,Blood Platelets ,Aging ,Platelet Aggregation ,Undefined ,STIM1 ,Filamins ,platelet function ,calcium entry ,030204 cardiovascular system & hematology ,Filamin ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Humans ,Platelet ,Calcium Signaling ,Calcium entry ,Chemistry ,Cell Biology ,Cell biology ,030104 developmental biology ,Gene Expression Regulation ,Calcium ,filamin A ,Function (biology) - Published
- 2018
32. STIM1 phosphorylation at Y
- Author
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Esther, Lopez, Irene, Frischauf, Isaac, Jardin, Isabella, Derler, Martin, Muik, Carlos, Cantonero, Gines M, Salido, Tarik, Smani, Juan A, Rosado, and Pedro C, Redondo
- Subjects
HEK293 Cells ,Intracellular Calcium-Sensing Proteins ,ORAI1 Protein ,Animals ,Humans ,Membrane Proteins ,Tyrosine ,Calcium ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Phosphorylation ,Calcium Release Activated Calcium Channels ,Neoplasm Proteins - Abstract
Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store-operated Ca
- Published
- 2018
33. Fluorescence-Based Measurements of the CRAC Channel Activity in Cell Populations
- Author
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Pedro C, Redondo, Alejandro, Berna-Erro, Natalia, Dionisio, and Juan A, Rosado
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Cytosol ,Spectrometry, Fluorescence ,Optical Imaging ,Animals ,Humans ,Calcium ,Calcium Signaling ,Flow Cytometry ,Calcium Release Activated Calcium Channels ,Ion Channel Gating ,Fluorescent Dyes ,Molecular Imaging - Abstract
Cytosolic Ca
- Published
- 2018
34. FKBP25 and FKBP38 regulate non-capacitative calcium entry through TRPC6
- Author
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Alejandro Berna-Erro, Esther Lopez, Ginés M. Salido, Pedro C. Redondo, and Juan A. Rosado
- Subjects
Platelets ,Blood Platelets ,Calmodulin ,FK506 ,TRPC6 ,FKBP38 ,Mice ,TRPC3 ,Immunophilins ,FKBP25 ,NCCE ,TRPC6 Cation Channel ,Animals ,Humans ,Molecular Biology ,Diacylglycerol kinase ,TRPC Cation Channels ,biology ,Chemistry ,Cell Biology ,Molecular biology ,Cell biology ,FKBP ,HEK293 Cells ,biology.protein ,Calcium ,Cell activation ,Intracellular ,Immunosuppressive Agents - Abstract
Non-capacitative calcium entry (NCCE) contributes to cell activation in response to the occupation of G protein-coupled membrane receptors. Thrombin administration to platelets evokes the synthesis of diacylglycerol downstream of PAR receptor activation. Diacylglycerol evokes NCCE through activating TRPC3 and TRPC6 in human platelets. Although it is known that immunophilins interact with TRPCs, the role of immunophilins in the regulation of NCCE remains unknown. Platelet incubation with FK506, an immunophilin antagonist, reduced OAG-evoked NCCE in a concentration-dependent manner, an effect that was independent on the inactivation of calcineurin (CaN). FK506 was unable to reduce NCCE evoked by OAG in platelets from TRPC6−/− mice. In HEK-293 cells overexpressing TRPC6, currents through TRPC6 were altered in the presence of FK506. We have found interaction between FKBP38 and other FKBPs, like FKBP25, FKBP12, and FKBP52 that were not affected by FK506, as well as with calmodulin (CaM). FK506 modified the pattern of association between FKBP25 and TRPCs as well as impaired OAG-evoked TRPC3 and TRPC6 coupling in both human and mouse platelets. By performing biotinylation experiments we have elucidated that FKBP25 and FKBP38 might be found at different cellular location, the plasma membrane and the already described intracellular locations. Finally, FKBP25 and FKBP38 silencing significantly inhibits OAG-evoked NCCE in MEG-01 and HEK293 cells, while overexpression of FKBP38 does not modify NCCE in HEK293 cells. All together, these findings provide strong evidence for a role of immunophilins, including FKBP25 and FKBP38, in NCCE mediated by TRPC6.
- Published
- 2015
- Full Text
- View/download PDF
35. Evaluation of the antiaggregant activity of ascorbyl phenolic esters with antioxidant properties
- Author
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Ginés M. Salido, Joaquín Altarejos, Juan A. Rosado, Pedro C. Redondo, Esther Lopez, Sofía Salido, and María del Carmen Ortega-Liébana
- Subjects
Blood Platelets ,Agonist ,endocrine system ,animal structures ,Antioxidant ,Platelet Aggregation ,Physiology ,medicine.drug_class ,animal diseases ,medicine.medical_treatment ,Drug Evaluation, Preclinical ,Ascorbic Acid ,Pharmacology ,Biochemistry ,Thrombin ,Phenols ,medicine ,Humans ,Platelet ,Calcium Signaling ,Platelet activation ,Phosphorylation ,Calcium metabolism ,Chemistry ,TOR Serine-Threonine Kinases ,Esters ,hemic and immune systems ,Free Radical Scavengers ,General Medicine ,Ascorbic acid ,eye diseases ,Platelet aggregation inhibitor ,Protein Processing, Post-Translational ,Platelet Aggregation Inhibitors ,medicine.drug - Abstract
Beneficial effects of the antioxidant L-ascorbic acid (Asc) in human health are well known. Its particular role in hemostasis deserves further consideration, since it has been described a dose-dependent effect of Asc in platelet activity. Contrary, it has been demonstrated that phenolic compounds have inhibitory effects on platelet aggregation stimulated by the physiological agonist thrombin (Thr). Here, we have evaluated the actions of three synthetic phenolic esters of Asc: L-ascorbyl 6-protocatechuate (Prot Asc), L-ascorbyl 6-gallate (Gal Asc), and L-ascorbyl 6-caffeate (Caf Asc). All these Asc derivatives exhibited greater radical scavenging activity than Asc, and in experiments using human platelets from healthy subjects, they do not evoke changes in platelet viability upon their administration. Nevertheless, these compounds altered platelet calcium homeostasis in response to Thr, although Prot Asc induced a smaller effect than Gal Asc, Caf Asc, and Asc. As a consequence, platelet aggregation was also impaired by these compounds, reporting Prot Asc and Caf Asc a weaker antiaggregant action than Gal Asc and Asc. Treatments with Gal Asc and Caf Asc altered in larger extent the phosphorylation pattern of pp60(Src) and mammalian target of rapamycin (mTOR) evoked by stimulating human platelets with Thr. Summarizing, Prot Asc is the ascorbyl phenolic ester with the strongest antioxidant properties and weakest antiaggregant actions, and its use as antioxidant may be safer than the rest of derivatives in order to prevent thrombotic alteration in patients that need treatment with antioxidant therapies.
- Published
- 2015
36. Role of mTOR1 and mTOR2 complexes in MEG-01 cell physiology
- Author
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María P. Granados, Juan A. Rosado, Ginés M. Salido, Javier J. López, Nuria Bermejo, José María Brull, Pedro C. Redondo, Esther Lopez, and Alejandro Berna-Erro
- Subjects
Blood Platelets ,0301 basic medicine ,Cell physiology ,Apoptosis ,Mechanistic Target of Rapamycin Complex 2 ,Tacrolimus Binding Protein 1A ,Mechanistic Target of Rapamycin Complex 1 ,Biology ,mTORC2 ,Cell Line ,Tacrolimus Binding Proteins ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Humans ,Transgenes ,Naphthyridines ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,Megakaryocyte Progenitor Cells ,Sirolimus ,Cell growth ,TOR Serine-Threonine Kinases ,RPTOR ,Cell Differentiation ,Hematology ,Cell biology ,030104 developmental biology ,Thrombopoietin ,Multiprotein Complexes ,030220 oncology & carcinogenesis ,Intracellular - Abstract
SummaryThe function of the mammalian target of rapamycin (mTOR) is upregulated in response to cell stimulation with growing and differentiating factors. Active mTOR controls cell proliferation, differentiation and death. Since mTOR associates with different proteins to form two functional macromolecular complexes, we aimed to investigate the role of the mTORI and mTOR2 complexes in MEG-01 cell physiology in response to thrombopoietin (TPO). By using mTOR antagonists and overexpressing FKBP38, we have explored the role of both mTOR complexes in proliferation, apoptosis, maturation-like mechanisms, endoplasmic reticulum-stress and the intracellular location of both active mTOR complexes during MEG-01 cell stimulation with TPO. The results demonstrate that mTOR1 and mTOR2 complexes play different roles in the physiology of MEG-01 cells and in the maturation-like mechanisms; hence, these findings might help to understand the mechanism underlying generation of platelets.
- Published
- 2015
37. Introduction: Overview of the Pathophysiological Implications of Store-Operated Calcium Entry in Mammalian Cells
- Author
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Juan A, Rosado
- Subjects
Mammals ,Animals ,Humans ,Calcium ,Calcium Channels ,Calcium Signaling - Abstract
Since store-operated Ca
- Published
- 2017
38. Cardiovascular and Hemostatic Disorders: SOCE and Ca
- Author
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Jose J, Lopez, Gines M, Salido, and Juan A, Rosado
- Subjects
Blood Platelets ,Hemostatic Disorders ,Cardiovascular Diseases ,Cell Membrane ,Animals ,Humans ,Calcium ,Calcium Channels ,Calcium Signaling ,Stromal Interaction Molecule 1 ,TRPC Cation Channels - Abstract
Among the Ca
- Published
- 2017
39. Filamin A Modulates Store-Operated Ca
- Author
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Jose J, Lopez, Letizia, Albarrán, Isaac, Jardín, Jose, Sanchez-Collado, Pedro C, Redondo, Nuria, Bermejo, Regis, Bobe, Tarik, Smani, and Juan A, Rosado
- Subjects
Blood Platelets ,Skin Neoplasms ,ORAI1 Protein ,Platelet Aggregation ,Filamins ,Cyclic AMP-Dependent Protein Kinases ,Neoplasm Proteins ,Cell Line, Tumor ,Serine ,Humans ,Calcium ,Protein Interaction Domains and Motifs ,Calcium Signaling ,Stromal Interaction Molecule 1 ,Phosphorylation ,Ion Channel Gating ,Melanoma ,Cytoskeleton ,Protein Binding - Abstract
Here, we provide evidence for the role of FLNA (filamin A) in the modulation of store-operated calcium entry (SOCE).SOCE is a major mechanism for calcium influx controlled by the intracellular CaOur results indicate that FLNA modulates SOCE and then the correct platelet function, by fine-tuning the distribution of STIM1 in the cytoskeleton and the interaction with Orai1 channels.
- Published
- 2017
40. Flavonoids and Platelet-Derived Thrombotic Disorders
- Author
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Raquel Diez-Bello, Pedro C. Redondo, Isaac Jardin, Juan A. Rosado, Mohammed El Haouari, Jose J. Lopez, Nieves Alonso, and S. Regodon
- Subjects
Cell signaling ,030204 cardiovascular system & hematology ,Pharmacology ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Drug Discovery ,Medicine ,Animals ,Humans ,Platelet ,Platelet activation ,030304 developmental biology ,Flavonoids ,0303 health sciences ,biology ,business.industry ,Kinase ,Organic Chemistry ,Cardiovascular Agents ,Thrombosis ,medicine.disease ,Platelet Activation ,Venous thrombosis ,Cardiovascular Diseases ,Mitogen-activated protein kinase ,Hemostasis ,biology.protein ,Molecular Medicine ,business - Abstract
Thrombotic disorders are characterized by an increase in the probability of the formation of unnecessary thrombi that might be due to the activation of the coagulation cascade or the circulating platelets. Platelets or thrombocytes play an essential role in hemostasis but abnormal platelet function leads to the development of a number of cardiovascular complications, including thrombotic disorders. Under pathological conditions, platelets are associated with the development of different thrombotic disorders, including atherosclerosis, arterial thrombosis and stroke, deep venous thrombosis and pulmonary embolism; therefore, platelets are the target of a number of anti-thrombotic strategies. Flavonoids, a large group of polyphenols ubiquitously expressed in fruits and vegetables that have attracted considerable attention because of their benefits in human health, including the reduction of the risk of cardiovascular disease. Flavonoids have been reported to reduce platelet activity by attenuating agonist-induced GPIIb/IIIa receptor activation, mobilization of intracellular free Ca2+, granule exocytosis, as well as activation of different signaling molecules such as mitogen- activated protein kinases or phospholipases. This review summarizes the current studies concerning the modulation of platelet activation by flavonoids, giving especial attention to those events associated to thrombotic disorders.
- Published
- 2017
41. Role of STIM2 in cell function and physiopathology
- Author
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Alejandro, Berna-Erro, Isaac, Jardin, Gines M, Salido, and Juan A, Rosado
- Subjects
Neoplasms ,Animals ,Humans ,Stromal Interaction Molecule 2 ,Topical Review ,Cardiovascular System ,Nervous System - Abstract
An endoplasmic reticulum (ER)‐resident protein that regulates cytosolic and ER free‐Ca2+ concentration by induction of store‐operated calcium entry: that is the original definition of STIM2 and its function. While its activity strongly depends on the amount of calcium stored in the ER, its function goes further, to intracellular signalling and gene expression. Initially under‐studied owing to the prominent function of STIM1, STIM2 came to be regarded as vital in mice, gradually emerging as an important player in the nervous system, and cooperating with STIM1 in the immune system. STIM2 has also been proposed as a relevant player in pathological conditions related to ageing, Alzheimer's and Huntington's diseases, autoimmune disorders and cancer. The discovery of additional functions, together with new splicing forms with opposite roles, has clarified existing controversies about STIM2 function in SOCE. With STIM2 being essential for life, but apparently not for development, newly available data demonstrate a complex and still intriguing behaviour that this review summarizes, updating current knowledge of STIM2 function.
- Published
- 2016
42. Transient receptor potential ankyrin-1 (TRPA1) modulates store-operated Ca2+ entry by regulation of STIM1-Orai1 association
- Author
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Ginés M. Salido, Letizia Albarran, Natalia Dionisio, Juan A. Rosado, Tarik Smani, and Jose J. Lopez
- Subjects
Blood Platelets ,Orai1 ,ORAI1 Protein ,STIM1 ,Nerve Tissue Proteins ,Biology ,TRPA1 ,Cell Line ,TRPC6 ,TRPC1 ,Transient receptor potential channel ,Transient Receptor Potential Channels ,Downregulation and upregulation ,Isothiocyanates ,TRP channel ,Humans ,Stromal Interaction Molecule 1 ,TRPA1 Cation Channel ,Molecular Biology ,ORAI1 ,Thrombin ,Membrane Proteins ,food and beverages ,Cell Differentiation ,MEG01 cell ,Cell Biology ,Neoplasm Proteins ,Cell biology ,rap GTP-Binding Proteins ,Purines ,Ca2+ entry ,Tetradecanoylphorbol Acetate ,Thapsigargin ,Acetanilides ,Calcium ,Calcium Channels ,Signal transduction ,Megakaryocytes ,Intracellular ,psychological phenomena and processes ,Protein Binding - Abstract
TRPA1 is a non-selective Ca(2+) permeable channel located in the plasma membrane that functions as a cellular sensor detecting mechanical, chemical and thermal stimuli, being a component of neuronal, epithelial, blood and smooth muscle tissues. TRPA1 has been shown to influence a broad range of physiological processes that involve Ca(2+)-dependent signaling pathways. Here we report that TRPA1 is expressed in MEG01 but not in platelets at the protein level. MEG01 cells maturation induced by PMA results in attenuation of TRPA1 protein expression and enhances thapsigargin-evoked Ca(2+) entry without altering the release of Ca(2+) from intracellular stores. Inhibition of TRPA1 by HC-030031 results in enhancement of both thrombin- and thapsigargin-stimulated Ca(2+) entry. Co-immunoprecipitation experiments revealed that TRPA1 associates with STIM1, as well as Orai1, TRPC1 and TRPC6. Downregulation of TRPA1 expression by MEG01 maturation, as well as pharmacological inhibition of TRPA1 by HC-030031, results in enhancement of the association between STIM1 and Orai1. Altogether, these findings provide evidence for a new and interesting function of TRPA1 in cellular function associated to the regulation of agonist-induced Ca(2+) entry by the modulation of STIM1/Orai1 interaction.
- Published
- 2013
- Full Text
- View/download PDF
43. The polybasic lysine-rich domain of plasma membrane-resident STIM1 is essential for the modulation of store-operated divalent cation entry by extracellular calcium
- Author
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Ginés M. Salido, Irene Frischauf, Geoffrey E. Woodard, Natalia Dionisio, Juan A. Rosado, Isaac Jardin, Alejandro Berna-Erro, and Jose J. Lopez
- Subjects
inorganic chemicals ,Thapsigargin ,ORAI1 Protein ,Cations, Divalent ,Gene Expression ,Divalent ,chemistry.chemical_compound ,Extracellular ,Humans ,Polylysine ,Stromal Interaction Molecule 1 ,Phosphorylation ,RNA, Small Interfering ,TRPC Cation Channels ,chemistry.chemical_classification ,Ion Transport ,ORAI1 ,Endoplasmic reticulum ,Cell Membrane ,Membrane Proteins ,STIM1 ,Cell Biology ,Neoplasm Proteins ,Protein Structure, Tertiary ,HEK293 Cells ,Amino Acid Substitution ,chemistry ,Biochemistry ,Phosphoserine ,Biophysics ,Calcium ,RNA Interference ,Calcium Channels ,Intracellular - Abstract
STIM1 acts as an endoplasmic reticulum Ca(2+) sensor that communicates the filling state of the intracellular stores to the store-operated channels. In addition, STIM1 is expressed in the plasma membrane, with the Ca(2+) binding EF-hand motif facing the extracellular medium; however, its role sensing extracellular Ca(2+) concentrations in store-operated Ca(2+) entry (SOCE), as well as the underlying mechanism remains unclear. Here we report that divalent cation entry stimulated by thapsigargin (TG) is attenuated by extracellular Ca(2+) in a concentration-dependent manner. Expression of the Ca(2+)-binding defective STIM1(D76A) mutant did not alter the surface expression of STIM1 but abolishes the regulation of divalent cation entry by extracellular Ca(2+). Orai1 and TRPC1 have been shown to play a major role in SOCE. Expression of the STIM1(D76A) mutant did not alter Orai1 phosphoserine content. TRPC1 silencing significantly attenuated TG-induced Mn(2+) entry. Expression of the STIM1(K684,685E) mutant impaired the association of plasma membrane STIM1 with TRPC1, as well as the regulation of TG-induced divalent cation entry by extracellular Ca(2+), which suggests that TRPC1 might be involved in the regulation of divalent cation entry by extracellular Ca(2+) mediated by plasma membrane-resident STIM1. Expression of the STIM1(D76A) or STIM1(K684,685E) mutants reduced store-operated divalent cation entry and resulted in loss of dependence on the extracellular Ca(2+) concentration, providing evidence for a functional role of plasma membrane-resident STIM1 in the regulation of store-operated divalent cation entry, which at least involves the EF-hand motif and the C-terminal polybasic lysine-rich domain.
- Published
- 2013
44. Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients
- Author
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Ginés M. Salido, Juan José Cubero, José María Brull, Alejandro Berna-Erro, Juan A. Rosado, Esther Lopez, Guadalupe Garcia Pino, Rocío Martinez, Pedro C. Redondo, Nuria Bermejo, and Raul Alvarado
- Subjects
Adult ,Blood Platelets ,Male ,Platelets ,medicine.medical_specialty ,Time Factors ,Platelet Aggregation ,Biology ,Internal medicine ,medicine ,Homeostasis ,Humans ,Platelet ,Everolimus ,Phosphorylation ,Protein Kinase Inhibitors ,thrombosis ,Kidney transplantation ,PI3K/AKT/mTOR pathway ,Aged ,Demography ,Sirolimus ,Calcium metabolism ,calcium ,rapamycin ,TOR Serine-Threonine Kinases ,Original Articles ,Cell Biology ,Middle Aged ,medicine.disease ,Kidney Transplantation ,Tacrolimus ,Enzyme Activation ,Endocrinology ,mTOR ,Molecular Medicine ,Female ,Signal Transduction ,medicine.drug - Abstract
The use of the mammal target of rapamycin (mTOR) inhibitors has been consolidated as the therapy of election for preventing graft rejection in kidney transplant patients, despite their immunosuppressive activity is less strong than anti-calcineurin agents like tacrolimus and cyclosporine A. Furthermore, as mTOR is widely expressed, rapamycin (a macrolide antibiotic produced by Streptomyces hygroscopicus) is recommended in patients presenting neoplasia due to its antiproliferative actions. Hence, we have investigated whether rapamycin presents side effects in the physiology of other cell types different from leucocytes, such as platelets. Blood samples were drawn from healthy volunteers and kidney transplant patients long-term medicated with rapamycin: sirolimus and everolimus. Platelets were either loaded with fura-2 or directly stimulated, and immunoassayed or fixed with Laemmli's buffer to perform the subsequent analysis of platelet physiology. Our results indicate that rapamycin evokes a biphasic time-dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers. Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia. All together our results show that long-term administration of rapamycin to kidney transplant patients evokes alteration in platelet function.
- Published
- 2013
45. Fine-tuning of microRNAs in Type 2 Diabetes Mellitus
- Author
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Raquel Diez-Bello, Ginés M. Salido, Juan A. Rosado, and Isaac Jardin
- Subjects
medicine.medical_specialty ,medicine.medical_treatment ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Insulin resistance ,Adipocyte ,Internal medicine ,Drug Discovery ,microRNA ,medicine ,Animals ,Humans ,Secretion ,030304 developmental biology ,Pharmacology ,0303 health sciences ,business.industry ,Insulin ,Organic Chemistry ,Type 2 Diabetes Mellitus ,medicine.disease ,Obesity ,MicroRNAs ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Diabetes Mellitus, Type 2 ,030220 oncology & carcinogenesis ,Molecular Medicine ,Pancreas ,business - Abstract
Type 2 diabetes mellitus is a metabolic disease widely spread across industrialized countries. Sedentary lifestyle and unhealthy alimentary habits lead to obesity, boosting both glucose and fatty acid in the bloodstream and eventually, insulin resistance, pancreas inflammation and faulty insulin production or secretion, all of them very well-defined hallmarks of type 2 diabetes mellitus. miRNAs are small sequences of non-coding RNA that may regulate several processes within the cells, fine-tuning protein expression, with an unexpected and subtle precision and in time-frames ranging from minutes to days. Since the discovery of miRNA and their possible implication in pathologies, several groups aimed to find a relationship between type 2 diabetes mellitus and miRNAs. Here we discuss the pattern of expression of different miRNAs in cultured cells, animal models and diabetic patients. We summarize the role of the most important miRNAs involved in pancreas growth and development, insulin secretion and liver, skeletal muscle or adipocyte insulin resistance in the context of type 2 diabetes mellitus.
- Published
- 2016
46. Second Messenger-Operated Calcium Entry Through TRPC6
- Author
-
Alexandre, Bouron, Sylvain, Chauvet, Stuart, Dryer, and Juan A, Rosado
- Subjects
Ion Transport ,TRPC6 Cation Channel ,Humans ,Calcium ,Second Messenger Systems ,TRPC Cation Channels - Abstract
Canonical transient receptor potential 6 (TRPC6) proteins assemble into heteromultimeric structures forming non-selective cation channels. In addition, many TRPC6-interacting proteins have been identified like some enzymes, channels, pumps, cytoskeleton-associated proteins, immunophilins, or cholesterol-binding proteins, indicating that TRPC6 are engaged into macromolecular complexes. Depending on the cell type and the experimental conditions used, TRPC6 activity has been reported to be controlled by diverse modalities. For instance, the second messenger diacylglycerol, store-depletion, the plant extract hyperforin or H2O2 have all been shown to trigger the opening of TRPC6 channels. A well-characterized consequence of TRPC6 activation is the elevation of the cytosolic concentration of Ca(2+). This latter response can reflect the entry of Ca(2+) through open TRPC6 channels but it can also be due to the Na(+)/Ca(2+) exchanger (operating in its reverse mode) or voltage-gated Ca(2+) channels (recruited in response to a TRPC6-mediated depolarization). Although TRPC6 controls a diverse array of biological functions in many tissues and cell types, its pathophysiological functions are far from being fully understood. This chapter covers some key features of TRPC6, with a special emphasis on their biological significance in kidney and blood cells.
- Published
- 2016
47. Historical Overview of Store-Operated Ca(2+) Entry
- Author
-
Letizia, Albarran, Jose J, Lopez, Ginés M, Salido, and Juan A, Rosado
- Subjects
Ion Transport ,Animals ,Humans ,Membrane Proteins ,Calcium ,TRPC Cation Channels - Abstract
Calcium influx is an essential mechanism for the activation of cellular functions both in excitable and non-excitable cells. In non-excitable cells, activation of phospholipase C by occupation of G protein-coupled receptors leads to the generation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which, in turn, initiate two Ca(2+) entry pathways: Ca(2+) release from intracellular Ca(2+) stores, signaled by IP3, leads to the activation of store-operated Ca(2+) entry (SOCE); on the other hand, DAG activates a distinct second messenger-operated pathway. SOCE is regulated by the filling state of the intracellular calcium stores. The search for the molecular components of SOCE has identified the stromal interaction molecule 1 (STIM1) as the Ca(2+) sensor in the endoplasmic reticulum and Orai1 as a store-operated channel (SOC) subunit. Furthermore, a number of reports have revealed that several members of the TRPC family of channels also take part of the SOC macromolecular complex. This introductory chapter summarizes the early pieces of evidence that led to the concept of SOCE and the components of the store-operated signaling pathway.
- Published
- 2016
48. Phospholipase A2 as a Molecular Determinant of Store-Operated Calcium Entry
- Author
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Tarik, Smani, Alejandro, Domínguez-Rodriguez, Paula, Callejo-García, Juan A, Rosado, and Javier, Avila-Medina
- Subjects
Phospholipases A2 ,Intracellular Calcium-Sensing Proteins ,Ion Transport ,Humans ,Membrane Proteins ,Calcium ,Calcium Signaling - Abstract
Activation of phospholipases A2 (PLA2) leads to the generation of biologically active lipid products that can affect numerous cellular events. Ca(2+)-independent PLA2 (iPLA2), also called group VI phospholipase A2, is one of the main types forming the superfamily of PLA2. Beside of its role in phospholipid remodeling, iPLA2 has been involved in intracellular Ca(2+) homeostasis regulation. Several studies proposed iPLA2 as an essential molecular player of store operated Ca(2+) entry (SOCE) in a large number of excitable and non-excitable cells. iPLA2 activation releases lysophosphatidyl products, which were suggested as agonists of store operated calcium channels (SOCC) and other TRP channels. Herein, we will review the important role of iPLA2 on the intracellular Ca(2+) handling focusing on its role in SOCE regulation and its implication in physiological and/or pathological processes.
- Published
- 2016
49. SARAF modulates TRPC1, but not TRPC6, channel function in a STIM1-independent manner
- Author
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Letizia Albarran, Luis Gómez, Jose J. Lopez, Juan A. Rosado, and Ginés M. Salido
- Subjects
inorganic chemicals ,0301 basic medicine ,Agonist ,medicine.drug_class ,Blotting, Western ,Stimulation ,Biology ,Biochemistry ,TRPC6 ,TRPC1 ,03 medical and health sciences ,0302 clinical medicine ,Intracellular Calcium-Sensing Proteins ,Cell Line, Tumor ,medicine ,TRPC6 Cation Channel ,Humans ,Immunoprecipitation ,Stromal Interaction Molecule 1 ,Receptor ,Molecular Biology ,TRPC ,TRPC Cation Channels ,ORAI1 ,Membrane Proteins ,STIM1 ,Biological Transport ,Cell Biology ,Cell biology ,Neoplasm Proteins ,030104 developmental biology ,Calcium ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Canonical transient receptor potential-1 (TRPC1) is an almost ubiquitously expressed channel that plays a relevant role in cell function. As other TRPC members, TRPC1 forms receptor-operated cation channels that exhibit both STIM1-dependent and store-independent behaviour. The STIM1 inhibitor SARAF (for store-operated Ca2+ entry (SOCE)-associated regulatory factor) modulates SOCE by interaction with the STIM1 region responsible for Orai1 activation (SOAR). Furthermore, SARAF modulates Ca2+ entry through the arachidonate-regulated Ca2+ (ARC) channels, consisting of Orai1 and Orai3 heteropentamers and plasma membrane-resident STIM1. While a role for STIM1–Orai1-mediated signals has been demonstrated, the possible role of SARAF in TRPC1 function remains unknown. Here, we provide evidence for the interaction of SARAF with TRPC1, independently of STIM1 both in STIM1-deficient NG115-401L cells and SH-SY5Y cells endogenously expressing STIM1. Silencing of SARAF expression in STIM1-deficient cells demonstrated that SARAF plays a negative regulatory role in TRPC1-mediated Ca2+ entry. The interaction of SARAF with TRPC1 in STIM1-deficient cells, as well as with the TRPC1 pool not associated with STIM1 in STIM1-expressing cells was enhanced by stimulation with the physiological agonist ATP. In contrast with TRPC1, we found that the interaction between SARAF and TRPC6 was constitutive rather than inducible by agonist stimulation. Furthermore, we found that SARAF expression silencing was without effect on Ca2+ entry evoked by agonists in TRPC6 overexpressing cells, as well as in Ca2+ influx evoked by the TRPC6 activator Hyp9. These findings provide evidence for a new regulator of TRPC1 channel function and highlight the relevance of SARAF in intracellular Ca2+ homeostasis.
- Published
- 2016
50. Dynamic interaction of SARAF with STIM1 and Orai1 to modulate store-operated calcium entry
- Author
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Jose J. Lopez, Alejandro Berna-Erro, Tarik Smani, Francisco E. Martín-Cano, Ginés M. Salido, Letizia Albarran, Nidhal Ben Amor, Juan A. Rosado, Ministerio de Economía y Competitividad (España), European Commission, Junta de Extremadura, and Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (España)
- Subjects
0301 basic medicine ,inorganic chemicals ,Thapsigargin ,ORAI1 Protein ,chemistry.chemical_element ,Calcium ,Biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Intracellular Calcium-Sensing Proteins ,Protein Interaction Mapping ,Homeostasis ,Humans ,Protein Interaction Maps ,Stromal Interaction Molecule 1 ,Calcium metabolism ,Multidisciplinary ,ORAI1 ,Membrane Proteins ,STIM1 ,Intracellular calcium-sensing proteins ,Store-operated calcium entry ,Cell biology ,Neoplasm Proteins ,030104 developmental biology ,chemistry ,Biochemistry ,030217 neurology & neurosurgery - Abstract
Ca2+ influx by store-operated Ca2+ channels is a major mechanism for intracellular Ca2+ homeostasis and cellular function. Here we present evidence for the dynamic interaction between the SOCE-associated regulatory factor (SARAF), STIM1 and Orai1. SARAF overexpression attenuated SOCE and the STIM1-Orai1 interaction in cells endogenously expressing STIM1 and Orai1 while RNAi-mediated SARAF silencing induced opposite effects. SARAF impaired the association between Orai1 and the Orai1-activating small fragment of STIM1 co-expressed in the STIM1-deficient NG115-401L cells. Cell treatment with thapsigargin or physiological agonists results in direct association of SARAF with Orai1. STIM1-independent interaction of SARAF with Orai1 leads to activation of this channel. In cells endogenously expressing STIM1 and Orai1, Ca2+ store depletion leads to dissociation of SARAF with STIM1 approximately 30s after treatment with thapsigargin, which paralleled the increase in SARAF-Orai1 interaction, followed by reinteraction with STIM1 and dissociation from Orai1. Co-expression of SARAF and either Orai1 or various N-terminal deletion Orai1 mutants did not alter SARAF-Orai1 interaction; however, expression of C-terminal deletion Orai1 mutants or blockade of the C-terminus of Orai1 impair the interaction with SARAF. These observations suggest that SARAF exerts an initial positive role in the activation of SOCE followed by the facilitation of SCDI of Orai1., Supported by MINECO (Grants BFU2013-45564-C2-1-P, BFU2013-45564-C2-2-P, BFU2011-24365 and RD12-0043-0016 (RETICEF)) and Junta de Extremadura-FEDER. JJL and LA are supported by Juan de la Cierva Program and MINECO fellowship BES-2011-043356, respectively.
- Published
- 2016
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