Publications by authors named "Gines M Salido"

153 Publications

Melatonin Induces Apoptosis and Modulates Cyclin Expression and MAPK Phosphorylation in Pancreatic Stellate Cells Subjected to Hypoxia.

Int J Mol Sci 2021 May 24;22(11). Epub 2021 May 24.

Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain.

In certain diseases of the pancreas, pancreatic stellate cells form an important part of fibrosis and are critical for the development of cancer cells. A hypoxic condition develops within the tumor, to which pancreatic stellate cells adapt and are able to proliferate. The consequence is the growth of the tumor. Melatonin, the product of the pineal gland, is gaining attention as an agent with therapeutic potential against pancreatic cancers. Its actions on tumor cells lead, in general, to a reduction in cell viability and proliferation. However, its effects on pancreatic stellate cells subjected to hypoxia are less known. In this study, we evaluated the actions of pharmacological concentrations of melatonin (1 mM-1 µM) on pancreatic stellate cells subjected to hypoxia. The results show that melatonin induced a decrease in cell viability at the highest concentrations tested. Similarly, the incorporation of BrdU into DNA was diminished by melatonin. The expression of cyclins A and D also was decreased in the presence of melatonin. Upon treatment of cells with melatonin, increases in the expression of major markers of ER stress, namely BIP, phospho-eIF2α and ATF-4, were detected. Modulation of apoptosis was noticed as an increase in caspase-3 activation. In addition, changes in the phosphorylated state of p44/42, p38 and JNK MAPKs were detected in cells treated with melatonin. A slight decrease in the content of α-smooth muscle actin was detected in cells treated with melatonin. Finally, treatment of cells with melatonin decreased the expression of matrix metalloproteinases 2, 3, 9 and 13. Our observations suggest that melatonin, at pharmacological concentrations, diminishes the proliferation of pancreatic stellate cells subjected to hypoxia through modulation of cell cycle, apoptosis and the activation of crucial MAPKs. Cellular responses might involve certain ER stress regulator proteins. In view of the results, melatonin could be taken into consideration as a potential therapeutic agent for pancreatic fibrosis.
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http://dx.doi.org/10.3390/ijms22115555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8197391PMC
May 2021

Melatonin Modulates the Antioxidant Defenses and the Expression of Proinflammatory Mediators in Pancreatic Stellate Cells Subjected to Hypoxia.

Antioxidants (Basel) 2021 Apr 8;10(4). Epub 2021 Apr 8.

Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain.

Pancreatic stellate cells (PSC) play a major role in the formation of fibrotic tissue in pancreatic tumors. On its side, melatonin is a putative therapeutic agent for pancreatic cancer and inflammation. In this work, the actions of melatonin on PSC subjected to hypoxia were evaluated. Reactive oxygen species (ROS) generation reduced (GSH) and oxidized (GSSG) levels of glutathione, and protein and lipid oxidation were analyzed. The phosphorylation of nuclear factor erythroid 2-related factor (Nrf2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and the regulatory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκBα) was studied. The expression of Nrf2-regulated antioxidant enzymes, superoxide dismutase (SOD) enzymes, cyclooxygenase 2 (COX-2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also studied. Total antioxidant capacity (TAC) was assayed. Finally, cell viability was studied. Under hypoxia and in the presence of melatonin generation of ROS was observed. No increases in the oxidation of proteins or lipids were detected. The phosphorylation of Nrf2 and the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1, heme oxygenase-1, SOD1, and of SOD2 were augmented. The TAC was increased. Protein kinase C was involved in the effects of melatonin. Melatonin decreased the GSH/GSSG ratio at the highest concentration tested. Cell viability dropped in the presence of melatonin. Finally, melatonin diminished the phosphorylation of NF-kB and the expression of COX-2, IL-6, and TNF-α. Our results indicate that melatonin, at pharmacological concentrations, modulates the red-ox state, viability, and the expression of proinflammatory mediators in PSC subjected to hypoxia.
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http://dx.doi.org/10.3390/antiox10040577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070371PMC
April 2021

Cross-Talk Between the Adenylyl Cyclase/cAMP Pathway and Ca Homeostasis.

Rev Physiol Biochem Pharmacol 2021 ;179:73-116

Department of Physiology and Institute of Molecular Pathology Biomarkers (IMPB), University of Extremadura, Cáceres, Spain.

Cyclic AMP and Ca are the first second or intracellular messengers identified, unveiling the cellular mechanisms activated by a plethora of extracellular signals, including hormones. Cyclic AMP generation is catalyzed by adenylyl cyclases (ACs), which convert ATP into cAMP and pyrophosphate. By the way, Ca, as energy, can neither be created nor be destroyed; Ca can only be transported, from one compartment to another, or chelated by a variety of Ca-binding molecules. The fine regulation of cytosolic concentrations of cAMP and free Ca is crucial in cell function and there is an intimate cross-talk between both messengers to fine-tune the cellular responses. Cancer is a multifactorial disease resulting from a combination of genetic and environmental factors. Frequent cases of cAMP and/or Ca homeostasis remodeling have been described in cancer cells. In those tumoral cells, cAMP and Ca signaling plays a crucial role in the development of hallmarks of cancer, including enhanced proliferation and migration, invasion, apoptosis resistance, or angiogenesis. This review summarizes the cross-talk between the ACs/cAMP and Ca intracellular pathways with special attention to the functional and reciprocal regulation between Orai1 and AC8 in normal and cancer cells.
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http://dx.doi.org/10.1007/112_2020_55DOI Listing
June 2021

Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple negative breast cancer cells.

J Biol Chem 2020 12 30:100254. Epub 2020 Dec 30.

Physiology, University of Extremadura, Spain.

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 non-tumoral 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.
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http://dx.doi.org/10.1074/jbc.RA120.015769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948746PMC
December 2020

PGRMC1 Inhibits Progesterone-Evoked Proliferation and Ca Entry Via STIM2 in MDA-MB-231 Cells.

Int J Mol Sci 2020 Oct 15;21(20). Epub 2020 Oct 15.

Department of Physiology (PHYCELL Group) of Veterinary Faculty and Institute of Molecular Pathology Biomarkers (IMPB) of University of Extremadura, 10003 Caceres, Spain.

Progesterone receptor membrane component 1 (PGRMC1) has been shown to regulate some cancer hallmarks. Progesterone (P) evokes intracellular calcium (Ca) 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 P-evoked Ca mobilization. Here, we found a new P-dependent Ca 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 Ca pathway, as evidenced by using siRNA STIM1. PGRMC1 silencing reduced the negative effect of P 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 P incubation for 48 h was enhanced in cells transfected with the small hairpin siRNA against PGRMC1 (shPGRMC1). These results provide evidence for a novel P-evoked Ca entry pathway that is downregulated by PGRMC1.
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http://dx.doi.org/10.3390/ijms21207641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589959PMC
October 2020

TRPC6 channel and its implications in breast cancer: an overview.

Biochim Biophys Acta Mol Cell Res 2020 12 18;1867(12):118828. Epub 2020 Aug 18.

Cellular Physiology Research Group, Department of Physiology, Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain.

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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http://dx.doi.org/10.1016/j.bbamcr.2020.118828DOI Listing
December 2020

Functional role of TRPC6 and STIM2 in cytosolic and endoplasmic reticulum Ca2+ content in resting estrogen receptor-positive breast cancer cells.

Biochem J 2020 09;477(17):3183-3197

Department of Physiology, Cell Physiology Research Group, University of Extremadura, 10003 Cáceres, Spain.

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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http://dx.doi.org/10.1042/BCJ20200560DOI Listing
September 2020

Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia.

Biol Cell 2020 Oct 12;112(10):280-299. Epub 2020 Jul 12.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain.

Background Information: Pancreatic stellate cells play a key role in the fibrosis that develops in diseases such as pancreatic cancer. In the growing tumour, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O ) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca concentration, mitochondrial free-Ca concentration and mitochondrial membrane potential were studied by fluorescence techniques. The status of enzymes responsible for the cellular oxidative state was analyzed by quantitative reverse transcription-polymerase chain reaction, high-performance liquid chromatography, spectrophotometric and fluorimetric methods and by Western blotting analysis. Cell viability and proliferation were studied by crystal violet test, 5-bromo-2-deoxyuridine cell proliferation test and Western blotting analysis. Finally, cell migration was studied employing the wound healing assay.

Results: Hypoxia induced an increase in intracellular and mitochondrial free-Ca concentration, whereas mitochondrial membrane potential was decreased. An increase in mitochondrial reactive oxygen species production was observed. Additionally, an increase in the oxidation of proteins and lipids was detected. Moreover, cellular total antioxidant capacity was decreased. Increases in the expression of superoxide dismutase 1 and 2 were observed and superoxide dismutase activity was augmented. Hypoxia evoked a decrease in the oxidized/reduced glutathione ratio. An increase in the phosphorylation of nuclear factor erythroid 2-related factor and in expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 were detected. The expression of cyclin A was decreased, whereas expression of cyclin D and the content of 5-bromo-2-deoxyuridine were increased. This was accompanied by an increase in cell viability. The phosphorylation state of c-Jun NH -terminal kinase was increased, whereas that of p44/42 and p38 was decreased. Finally, cells subjected to hypoxia maintained migration ability.

Conclusions And Significance: Hypoxia creates pro-oxidant conditions in pancreatic stellate cells to which cells adapt and leads to increased viability and proliferation.
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http://dx.doi.org/10.1111/boc.202000020DOI Listing
October 2020

The melatonin receptor antagonist luzindole induces the activation of cellular stress responses and decreases viability of rat pancreatic stellate cells.

J Appl Toxicol 2020 11 22;40(11):1554-1565. Epub 2020 Jun 22.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain.

In this study, we have examined the effects of luzindole, a melatonin receptor-antagonist, on cultured pancreatic stellate cells. Intracellular free-Ca concentration, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPK), endoplasmic reticulum stress and cell viability were analyzed. Stimulation of cells with the luzindole (1, 5, 10 and 50 μm) evoked a slow and progressive increase in intracellular free Ca ([Ca ] ) towards a plateau. The effect of the compound on Ca mobilization depended on the concentration used. Incubation of cells with the sarcoendoplasmic reticulum Ca -ATPase inhibitor thapsigargin (1 μm), in the absence of Ca in the extracellular medium, induced a transient increase in [Ca ] . In the presence of thapsigargin, the addition of luzindole to the cells failed to induce further mobilization of Ca . Luzindole induced a concentration-dependent increase in ROS generation, both in the cytosol and in the mitochondria. This effect was smaller in the absence of extracellular Ca . In the presence of luzindole the phosphorylation of p44/42 and p38 MAPKs was increased, whereas no changes in the phosphorylation of JNK could be noted. Moreover, the detection of the endoplasmic reticulum stress-sensor BiP was increased in the presence of luzindole. Finally, viability was decreased in cells treated with luzindole. Because cellular membrane receptors for melatonin have not been detected in pancreatic stellate cells, we conclude that luzindole could exert direct effects that are not mediated through its action on melatonin membrane receptors.
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http://dx.doi.org/10.1002/jat.4018DOI Listing
November 2020

Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry.

Int J Mol Sci 2020 May 7;21(9). Epub 2020 May 7.

Department of Physiology, Faculty of Veterinary Medicine and Institute of Molecular Pathology Biomarkers (IMPB), University of Extremadura, 10003 Cáceres, Spain.

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 Ca entry through the arachidonate-regulated Ca-selective channels (ARC channels). Here, the analysis of the changes in the intracellular Ca homeostasis revealed that, despite MDA-MB-231 cells expressing the ARC channel components Orai1, Orai3, and STIM1, AA does not evoke Ca entry in these cells. We observed that AA evokes Ca 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 Ca influx via ARC channels.
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http://dx.doi.org/10.3390/ijms21093315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247542PMC
May 2020

Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression.

J Physiol Biochem 2020 May 2;76(2):345-355. Epub 2020 May 2.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Avenida de las Ciencias s/n, E-10003, Cáceres, Spain.

In this study, the effects of melatonin (1 μM-1 mM) on pancreatic stellate cells (PSC) have been examined. Cell viability and proliferation, caspase-3 activation, and the expression of cyclin A and cyclin D were analyzed. Our results show that melatonin decreased PSC viability in a time- and concentration-dependent manner. This effect was not inhibited by treatment of cells with MT1, MT2, calmodulin, or ROR-alpha inhibitors prior to melatonin addition. Activation of caspase-3 in response to melatonin was detected. The expression of cyclin A and cyclin D was decreased in cells treated with melatonin. Finally, changes in BrdU incorporation into the newly synthesized DNA of proliferating cells were also observed in the presence of melatonin. We conclude that melatonin, at pharmacological concentrations, modulates proliferation of PSC through activation of apoptosis and involving crucial regulators of the cell cycle. These actions might not require specific melatonin receptors. Our observations suggest that melatonin, at high doses, could potentially exert anti-fibrotic effects and, thus, could be taken into consideration as supportive treatment in the therapy of pancreatic diseases.
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http://dx.doi.org/10.1007/s13105-020-00740-6DOI Listing
May 2020

Melatonin modulates red-ox state and decreases viability of rat pancreatic stellate cells.

Sci Rep 2020 04 14;10(1):6352. Epub 2020 Apr 14.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain.

In this work we have studied the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSC). Cell viability was analyzed by AlamarBlue test. Production of reactive oxygen species (ROS) was monitored following CM-HDCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψ) was monitored by TMRM-derived fluorescence. Reduced (GSH) and oxidized (GSSG) levels of glutathione were determined by fluorescence techniques. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Determination of SOD activity and total antioxidant capacity (TAC) were carried out by colorimetric methods, whereas expression of SOD was analyzed by Western blotting and RT-qPCR. The results show that melatonin decreased PSC viability in a concentration-dependent manner. Melatonin evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Oxidation of proteins was detected in the presence of melatonin, whereas lipids oxidation was not observed. Depolarization of ψ was noted with 1 mM melatonin. A decrease in the GSH/GSSG ratio was observed, that depended on the concentration of melatonin used. A concentration-dependent increase in the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 was detected in cells incubated with melatonin. Finally, decreases in the expression and in the activity of superoxide dismutase were observed. We conclude that pharmacological concentrations melatonin modify the redox state of PSC, which might decrease cellular viability.
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http://dx.doi.org/10.1038/s41598-020-63433-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156707PMC
April 2020

TRPC Channels in the SOCE Scenario.

Cells 2020 01 5;9(1). Epub 2020 Jan 5.

Department of Physiology (Cell Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003-Caceres, Spain.

Transient receptor potential (TRP) proteins form non-selective Ca 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 , it is well known that these channels are activated by stimuli that induce PIP hydrolysis. The canonical TRP (TRPC) channels have long been suggested to be constituents of the store-operated Ca (SOC) channels; however, none of the TRPC channels generate Ca currents that resemble . STIM1 and Orai1 have been identified as the components of the Ca release-activated Ca (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 . STIM1 has been found to interact to and activate Orai1 and TRPC1 by different mechanisms and the involvement of TRPC1 in store-operated Ca entry requires both STIM1 and Orai1. In addition to the participation of TRPC1 in the 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.
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http://dx.doi.org/10.3390/cells9010126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016597PMC
January 2020

Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells.

Cancers (Basel) 2019 Oct 23;11(11). Epub 2019 Oct 23.

Department of Physiology, (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain.

Orai1 plays a major role in store-operated Ca entry (SOCE) in triple-negative breast cancer (TNBC) cells. This channel is inactivated via different mechanisms, including protein kinase C (PKC) and protein kinase A (PKA)-dependent phosphorylation at Ser-27 and Ser-30 or Ser-34, respectively, which shapes the Ca responses to agonists. The Ca calmodulin-activated adenylyl cyclase type 8 (AC8) was reported to interact directly with Orai1, thus mediating a dynamic interplay between the Ca- and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Here, we show that the breast cancer cell lines MCF7 and MDA-MB-231 exhibit enhanced expression of Orai1 and AC8 as compared to the non-tumoral breast epithelial MCF10A cell line. In these cells, AC8 interacts with the Orai1α variant in a manner that is not regulated by Orai1 phosphorylation. AC8 knockdown in MDA-MB-231 cells, using two different small interfering RNAs (siRNAs), attenuates thapsigargin (TG)-induced Ca entry and also Ca influx mediated by co-expression of Orai1 and the Orai1-activating small fragment (OASF) of STIM1 (stromal interaction molecule-1). Conversely, AC8 overexpression enhances SOCE, as well as Ca entry, in cells co-expressing Orai1 and OASF. In MDA-MB-231 cells, we found that AC8 overexpression reduces the Orai1 phosphoserine content, thus suggesting that AC8 interferes with Orai1 serine phosphorylation, which takes place at residues located in the AC8-binding site. Consistent with this, the subset of Orai1 associated with AC8 in naïve MDA-MB-231 cells is not phosphorylated in serine residues in contrast to the AC8-independent Orai1 subset. AC8 expression knockdown attenuates migration of MCF7 and MDA-MB-231 cells, while this maneuver has no effect in the MCF10A cell line, which is likely attributed to the low expression of AC8 in these cells. We found that AC8 is required for FAK (focal adhesion kinase) phosphorylation in MDA-MB-231 cells, which might explain its role in cell migration. Finally, we found that AC8 is required for TNBC cell proliferation. These findings indicate that overexpression of AC8 in breast cancer MDA-MB-231 cells impairs the phosphorylation-dependent Orai1 inactivation, a mechanism that might support the enhanced ability of these cells to migrate.
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http://dx.doi.org/10.3390/cancers11111624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893434PMC
October 2019

Molecular Basis and Regulation of Store-Operated Calcium Entry.

Adv Exp Med Biol 2020 ;1131:445-469

Department of Physiology, Cell Physiology Research Group and Institute of Molecular Pathology Biomarkers, University of Extremadura, Cáceres, Spain.

Store-operated Ca entry (SOCE) is a ubiquitous mechanism for Ca influx in mammalian cells with important physiological implications. Since the discovery of SOCE more than three decades ago, the mechanism that communicates the information about the amount of Ca accumulated in the intracellular Ca stores to the plasma membrane channels and the nature of these channels have been matters of intense investigation and debate. The stromal interaction molecule-1 (STIM1) has been identified as the Ca sensor of the intracellular Ca compartments that activates the store-operated channels. STIM1 regulates two types of store-dependent channels: the Ca release-activated Ca (CRAC) channels, formed by Orai1 subunits, that conduct the highly Ca selective current I and the cation permeable store-operated Ca (SOC) channels, which consist of Orai1 and TRPC1 proteins and conduct the non-selective current I . While the crystal structure of Drosophila CRAC channel has already been solved, the architecture of the SOC channels still remains unclear. The dynamic interaction of STIM1 with the store-operated channels is modulated by a number of proteins that either support the formation of the functional STIM1-channel complex or protect the cell against Ca overload.
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http://dx.doi.org/10.1007/978-3-030-12457-1_17DOI Listing
October 2019

The melatonin receptor antagonist luzindole induces Ca mobilization, reactive oxygen species generation and impairs trypsin secretion in mouse pancreatic acinar cells.

Biochim Biophys Acta Gen Subj 2019 11 2;1863(11):129407. Epub 2019 Aug 2.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain. Electronic address:

Background: In this work we studied the effects of the melatonin receptor-antagonist luzindole (1 μM-50 μM) on isolated mouse pancreatic acinar cells.

Methods: Changes in intracellular free-Ca concentration, reactive oxygen species production and trypsin secretion were analyzed.

Results: Luzindole induced increases in [Ca] that diminished CCK-8 induced Ca mobilization, compared with that observed when CCK-8 was applied alone. Treatment of cells with thapsigargin (1 μM), in the absence of Ca in the extracellular medium, evoked a transient increase in [Ca]. The additional incubation of cells with luzindole (10 μM) failed to induce further mobilization of Ca. In the presence of luzindole a concentration-dependent increase in ROS generation was observed that decreased in the absence of Ca or by pretreatment of cells with melatonin (100 μM). Incubation of pancreatic acinar cells with luzindole (10 μM) impaired CCK-8-induced trypsin secretion. Melatonin was unable to revert the effect of luzindole on CCK-8-induced trypsin secretion.

Conclusion: The melatonin receptor-inhibitor luzindole induces Ca-mediated pro-oxidative conditions and impairment of enzyme secretion, which creates a situation in pancreatic acinar cells that might compromise their function.

General Significance: The effects of luzindole that we have observed, might be unspecific and could mislead the observations when it is used to study the actions of melatonin on the gland. Another possibility is that melatonin receptors exhibit a basal or agonist-independent activity in pancreatic acinar cells, which might be modulated by melatonin or luzindole.
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http://dx.doi.org/10.1016/j.bbagen.2019.07.016DOI Listing
November 2019

STIM1 phosphorylation at Y modulates its interaction with SARAF and the activation of SOCE and .

J Cell Sci 2019 05 15;132(10). Epub 2019 May 15.

Department of Physiology, Cell Physiology Research Group, Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain

Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store-operated Ca 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 Y 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 Ca release-activated Ca current (). 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 Ca store depletion. Expression of the STIM1 Y316F mutant enhanced slow Ca-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 Y plays a relevant functional role in the activation and modulation of SOCE.
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http://dx.doi.org/10.1242/jcs.226019DOI Listing
May 2019

Melatonin induces reactive oxygen species generation and changes in glutathione levels and reduces viability in human pancreatic stellate cells.

J Physiol Biochem 2019 Jun 13;75(2):185-197. Epub 2019 Mar 13.

Department of Physiology, Institute of Molecular Pathology Biomarkers, University of Extremadura, Avenida Universidad s/n, 10003, Cáceres, Spain.

In this study, the effects of pharmacological concentrations of melatonin (1 μM-1 mM) on human pancreatic stellate cells (HPSCs) have been examined. Cell type-specific markers and expression of melatonin receptors were analyzed by western blot analysis. Changes in intracellular free Ca concentration were followed by fluorimetric analysis of fura-2-loaded cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined by fluorescence techniques. Production of reactive oxygen species (ROS) was monitored following 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester and MitoSOX™ Red-derived fluorescence. Cell viability was studied using the AlamarBlue test. Cultured cells expressed markers typical of stellate cells. However, cell membrane receptors for melatonin could not be detected. Thapsigargin, bradykinin, or melatonin induced changes in intracellular free Ca concentration. In the presence of the indole, a decrease in the GSH/GSSG ratio was observed that depended on the concentration of melatonin used. Furthermore, the indole evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Finally, melatonin decreased HPSC viability in a time and concentration-dependent manner. We conclude that melatonin, at pharmacological concentrations, induces changes in the oxidative state of HPSC. This might regulate cellular viability and could not involve specific plasma membrane receptors.
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http://dx.doi.org/10.1007/s13105-019-00671-xDOI Listing
June 2019

Store-Operated Ca Entry in Breast Cancer Cells: Remodeling and Functional Role.

Int J Mol Sci 2018 Dec 14;19(12). Epub 2018 Dec 14.

Department of Physiology, (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain.

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. Ca influx from the extracellular medium, but more specifically store-operated Ca 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.
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http://dx.doi.org/10.3390/ijms19124053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321005PMC
December 2018

EFHB is a Novel Cytosolic Ca2+ Sensor That Modulates STIM1-SARAF Interaction.

Cell Physiol Biochem 2018 27;51(3):1164-1178. Epub 2018 Nov 27.

Department of Physiology, (Cellular Physiology and Muscle Physiology Research Groups), Institute of Molecular Pathology Biomarkers, University of Extremadura, Cáceres, Spain.

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.
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http://dx.doi.org/10.1159/000495494DOI Listing
January 2019

TRPC6 Channels Are Required for Proliferation, Migration and Invasion of Breast Cancer Cell Lines by Modulation of Orai1 and Orai3 Surface Exposure.

Cancers (Basel) 2018 Sep 14;10(9). Epub 2018 Sep 14.

Cellular Physiology Research Group, Department of Physiology, Institute of Molecular Pathology Biomarkers, University of Extremadura, 10003 Caceres, Spain.

Transient receptor potential channels convey signaling information from a number of stimuli to a wide variety of cellular functions, mainly by inducing changes in cytosolic Ca concentration. Different members of the TRPC, TRPM and TRPV subfamilies have been reported to play a role in tumorigenesis. Here we show that the estrogen receptor positive and triple negative breast cancer cell lines, MCF7 and MDA-MB-231, respectively, exhibit enhanced expression of the TRPC6 channel as compared to the non-tumoral MCF10A cell line. In vitro TRPC6 knockdown using shRNA impaired MCF7 and MDA-MB-231 cell proliferation, migration and invasion detected by BrdU incorporation, wound healing and Boyden chamber assays, respectively. Using RNAi-mediated TRPC6 silencing as well as overexpression of the pore-dead dominant-negative TRPC6 mutant we have found that TRPC6 plays a relevant role in the activation of store-operated Ca entry in the breast cancer cell lines but not in non-tumoral breast cells. Finally, we have found that TRPC6 interacts with Orai1 and Orai3 in MCF7 and MDA-MB-231 cells and is required for the translocation of Orai1 and Orai3 to the plasma membrane in MDA-MB-231 and MCF7 cells, respectively, upon Ca store depletion. These findings introduce a novel mechanism for the modulation of Ca influx and the development of different cancer hallmarks in breast cancer cells.
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http://dx.doi.org/10.3390/cancers10090331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162527PMC
September 2018

Sulfanilic acid increases intracellular free-calcium concentration, induces reactive oxygen species production and impairs trypsin secretion in pancreatic AR42J cells.

Food Chem Toxicol 2018 Oct 3;120:71-80. Epub 2018 Jul 3.

Institute of Molecular Pathology Biomarkers, University of Extremadura, Caceres, Spain. Electronic address:

We studied the effects of the tartrazine-metabolite sulfanilic acid on the physiology of pancreatic AR42J cells. Sulfanilic acid (1 μM-1 mM) induced a slow and progressive increase in intracellular free-calcium concentration that reached a plateau. The effect of sulfanilic acid was not concentration-dependent. Stimulation of cells with thapsigargin (1 μM) after treatment with sulfanilic acid (1 mM) induced a smaller Ca response compared with that obtained with thapsigargin alone. Sulfanilic acid induced a concentration-dependent production of reactive oxygen species; however, this effect was not Ca-dependent. Depolarization of mitochondrial membrane potential was observed at the concentration of 1 mM sulfanilic acid. In the presence of the compound a decrease in the GSH/GSSG ratio was observed. A decrease in the expression of superoxide dismutase 2 was noted. Finally, stimulation of cells with CCK-8 led to a concentration-dependent increase of trypsin secretion that was impaired by pretreatment of cells with sulfanilic acid. Preincubation of cells with the antioxidant melatonin (100 μM) reduced the effect of sulfanilic acid on trypsin secretion. We conclude that sulfanilic acid might induce oxidative stress, which could alter Ca signaling and enzyme secretion in pancreatic AR42J cells. This creates a situation potentially leading to damage of the exocrine pancreas.
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http://dx.doi.org/10.1016/j.fct.2018.07.001DOI Listing
October 2018

Stanniocalcin 2 Regulates Non-capacitative Ca Entry and Aggregation in Mouse Platelets.

Front Physiol 2018 23;9:266. Epub 2018 Mar 23.

Department of Physiology (PHYCELL) of the Veterinary Faculty, University of Extremadura, Cáceres, Spain.

Stanniocalcin 2 (STC2) is a fish protein that controls body Ca and phosphate metabolism. STC2 has also been described in mammals, and as platelet function highly depends on both extracellular and intracellular Ca, we have explored its expression and function in these cells. STC2 mice exhibit shorter tail bleeding time than WT mice. Platelets from STC2-deficient mice showed enhanced aggregation, as well as enhanced Ca mobilization in response to the physiological agonist thrombin (Thr) and the diacylglycerol analog, OAG, a selective activator of the non-capacitative Ca entry channels. Interestingly, platelets from STC2 mice exhibit attenuated interaction between STIM1 and Orai1 in response to Thr, thus suggesting that STC2 is required for Thr-evoked STIM1-Orai1 interaction and the subsequent store-operated Ca entry (SOCE). We have further assessed possible changes in the expression of the most relevant channels involved in non-capacitative Ca entry in platelets. Then, protein expression of Orai3, TRPC3 and TRPC6 were evaluated by Western blotting, and the results revealed that while the expression of Orai3 was enhanced in the STC2-deficient mice, others like TRPC3 and TRPC6 remains almost unaltered. Summarizing, our results provide for the first time evidence for a role of STC2 in platelet physiology through the regulation of agonist-induced Ca entry, which might be mediated by the regulation of Orai3 channel expression.
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http://dx.doi.org/10.3389/fphys.2018.00266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876523PMC
March 2018

Fine-tuning of store-operated calcium entry by fast and slow Ca-dependent inactivation: Involvement of SARAF.

Biochim Biophys Acta Mol Cell Res 2018 Mar 6;1865(3):463-469. Epub 2017 Dec 6.

Department of Physiology (Cell Physiology Research Group), University of Extremadura, Cáceres, Spain.

Store-operated Ca entry (SOCE) is a functionally relevant mechanism for Ca influx present in electrically excitable and non-excitable cells. Regulation of Ca entry through store-operated channels is essential to maintain an appropriate intracellular Ca homeostasis and prevent cell damage. Calcium-release activated channels exhibit Ca-dependent inactivation mediated by two temporally separated mechanisms: fast Ca-dependent inactivation takes effect in the order of milliseconds and involves the interaction of Ca with residues in the channel pore while slow Ca-dependent inactivation (SCDI) develops over tens of seconds, requires a global rise in [Ca] and is a mechanism regulated by mitochondria. Recent studies have provided evidence that the protein SARAF (SOCE-associated regulatory factor) is involved in the mechanism underlying SCDI of Orai1. SARAF is an endoplasmic reticulum (ER) membrane protein that associates with STIM1 and translocate to plasma membrane-ER junctions in a STIM1-dependent manner upon store depletion to modulate SOCE. SCDI mediated by SARAF depends on the location of the STIM1-Orai1 complex within a PI(4,5)P-rich microdomain. SARAF also interacts with Orai1 and TRPC1 in cells endogenously expressing STIM1 and cells with a low STIM1 expression and modulates channel function. This review focuses on the modulation by SARAF of SOCE and other forms of Ca influx mediated by Orai1 and TRPC1 in order to provide spatio-temporally regulated Ca signals.
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http://dx.doi.org/10.1016/j.bbamcr.2017.12.001DOI Listing
March 2018

Fine-tuning of microRNAs in Type 2 Diabetes Mellitus.

Curr Med Chem 2019 ;26(22):4102-4118

Institute of Molecular Pathology Biomarkers & Department of Physiology (Cell Physiology Research Group), University of Extremadura, 10003-Caceres, Spain.

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.
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http://dx.doi.org/10.2174/0929867325666171205163944DOI Listing
November 2019

Cardiovascular and Hemostatic Disorders: SOCE and Ca Handling in Platelet Dysfunction.

Adv Exp Med Biol 2017 ;993:453-472

Cell Physiology Research Group, Department of Physiology, University of Extremadura, Cáceres, Spain.

Among the Ca entry mechanisms in platelets, store-operated Ca entry (SOCE) plays a prominent role as it is necessary to achieve full activation of platelet functions and replenish intracellular Ca stores. In platelets, as in other non-excitable cells, SOCE has been reported to involve the activation of plasma membrane channels by the ER Ca sensor STIM1. Despite electrophysiological studies are not possible in human platelets, indirect analyses have revealed that the Ca-permeable channels involve Orai1 and, most likely, TRPC1 subunits. A relevant role for the latter has not been found in mouse platelets. There is a body of evidence revealing a number of abnormalities in SOCE or in its molecular regulators that result in qualitative platelet disorders and, as a consequence, altered platelet responsiveness upon stimulation with multiple physiological agonists. Platelet SOCE abnormalities include STIM1 and Orai1 mutations. This chapter summarizes the current knowledge in this field, as well as the disorders associated to platelet SOCE dysfunction.
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http://dx.doi.org/10.1007/978-3-319-57732-6_23DOI Listing
December 2017

Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

J Cell Biochem 2018 01 5;119(1):1122-1133. Epub 2017 Oct 5.

Department of Physiology (Cell Physiology Research Group), University of Extremadura, Caceres, Spain.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca concentration ([Ca ] ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca ] . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells.
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http://dx.doi.org/10.1002/jcb.26280DOI Listing
January 2018

TRPs in Pain Sensation.

Front Physiol 2017 9;8:392. Epub 2017 Jun 9.

Cell Physiology Research Group, Department of Physiology, University of ExtremaduraCáceres, Spain.

According to the International Association for the Study of Pain (IASP) pain is characterized as an "unpleasant sensory and emotional experience associated with actual or potential tissue damage". The TRP super-family, compressing up to 28 isoforms in mammals, mediates a myriad of physiological and pathophysiological processes, pain among them. TRP channel might be constituted by similar or different TRP subunits, which will result in the formation of homomeric or heteromeric channels with distinct properties and functions. In this review we will discuss about the function of TRPs in pain, focusing on TRP channles that participate in the transduction of noxious sensation, especially TRPV1 and TRPA1, their expression in nociceptors and their sensitivity to a large number of physical and chemical stimuli.
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http://dx.doi.org/10.3389/fphys.2017.00392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465271PMC
June 2017

Role of STIM2 in cell function and physiopathology.

J Physiol 2017 05 19;595(10):3111-3128. Epub 2017 Feb 19.

Department of Physiology (Cell Physiology Research Group), University of Extremadura, 10003, Cáceres, Spain.

An endoplasmic reticulum (ER)-resident protein that regulates cytosolic and ER free-Ca 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.
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http://dx.doi.org/10.1113/JP273889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430229PMC
May 2017

SARAF modulates TRPC1, but not TRPC6, channel function in a STIM1-independent manner.

Biochem J 2016 10 9;473(20):3581-3595. Epub 2016 Aug 9.

Department of Physiology (Cellular Physiology Research Group), University of Extremadura, Cáceres 10003, Spain.

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 Ca entry (SOCE)-associated regulatory factor) modulates SOCE by interaction with the STIM1 region responsible for Orai1 activation (SOAR). Furthermore, SARAF modulates Ca entry through the arachidonate-regulated Ca (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 Ca 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 Ca entry evoked by agonists in TRPC6 overexpressing cells, as well as in Ca 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 Ca homeostasis.
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http://dx.doi.org/10.1042/BCJ20160348DOI Listing
October 2016