Juan Rosado | Universidad de Extremadura (original) (raw)

Papers by Juan Rosado

Research paper thumbnail of Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

Cellular and Molecular Life Sciences

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-acti... more 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 plas...

Research paper thumbnail of prodomain ppFurin enhances SOCE through TRPC 6 activation in 2 breast cancer cells 3 4

The intracellular calcium concentration ([Ca 2+ ]i) modulation plays a key role in the regulation... more The intracellular calcium concentration ([Ca 2+ ]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca 2+ ]i homeostasis is involved in tumor initiation and progression. Here we show that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced SOCE through TRPC6 activation that associated reduced cells malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments, revealed that the ppFurin-expressing cells are unable to process adequately the PC substrates proVEGF-C and proIGF-1R. Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 significantly induces Ca 2+ entry which is impaired by silencing of TRPC6 expression. Analysis of TRPC6 activation revealed its up-regulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. The expression of ppFurin in MDA-MB-231 cells reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca 2+ mobilization leading to breast cancer cells malignant phenotype repression and reduction of their resistance to treatments.

Research paper thumbnail of Protein and Vesicle Trafficking , Cytoskeleton Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca 2 entry in mouse pancreatic acinar cells

Rosado, Juan A., Pedro C. Redondo, Ginés M. Salido, Stewart O. Sage, and Jose A. Pariente. Cleava... more Rosado, Juan A., Pedro C. Redondo, Ginés M. Salido, Stewart O. Sage, and Jose A. Pariente. Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca entry in mouse pancreatic acinar cells. Am J Physiol Cell Physiol 288: C214–C221, 2005. First published September 8; doi:10.1152/ajpcell.00241.2004.—We recently reported that store-operated Ca entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as “secretion-like coupling.” As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosomeassociated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stim...

Research paper thumbnail of Role of Orai3 in the Pathophysiology of Cancer

International Journal of Molecular Sciences

The mammalian exclusive Orai3 channel participates in the generation and/or modulation of two ind... more 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 ass...

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

Biochemical Journal

TRPC6 forms non-selective cation channels activated by a variety of stimuli that are involved in ... more 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 cytosol...

Research paper thumbnail of Furin Prodomain ppFurin Enhances Ca2+ Entry Through Orai and TRPC6 Channels’ Activation in Breast Cancer Cells

Cancers

The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation o... more The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we showed that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced store-operated calcium entry (SOCE) and reduced the cell malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments revealed that the ppFurin-expressing cells are unable to adequately process the proprotein convertase (PC) substrates vascular endothelial growth factor C (proVEGF-C) and insulin-like growth factor-1 receptor (proIGF-1R). Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 and BT20 cells significantly enhanced SOCE...

Research paper thumbnail of PGRMC1 Inhibits Progesterone-Evoked Proliferation and Ca2+ Entry Via STIM2 in MDA-MB-231 Cells

International Journal of Molecular Sciences

Progesterone receptor membrane component 1 (PGRMC1) has been shown to regulate some cancer hallma... more 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...

Research paper thumbnail of Experiencias de Docencia Virtual en Facultades de Medicina Españolas durante la pandemia COVID-19 (I): Anatomía, Fisiología, Fisiopatología, Oncología

Revista Española de Educación Médica

Presentamos un resumen de las actividades que algunos profesores de Facultades de Medicina españo... more Presentamos un resumen de las actividades que algunos profesores de Facultades de Medicina españolas han llevado a cabo durante las 3 semanas previas a las vacaciones de primavera. En éstas, la docencia presencial tuvo que ser sustituída por actividades en línea o virtuales, a causa de la implantación del estado de alarma en España, que motivó el cierre completo de las Universidades desde el 13 de marzo de 2020. We present a summary of the activities that some professors of Schools of Medicine of Spain have carried out during the 3 weeks prior to spring break. In these, face-to-face teaching had to be replaced by online or virtual activities, due to the implementation of the state of alarm in Spain, which led to the complete closure of the Universities since March 13, 2020.

Research paper thumbnail of Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry

International Journal of Molecular Sciences

Arachidonic acid (AA) is a phospholipase A2 metabolite that has been reported to mediate a pletho... more 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 eff...

Research paper thumbnail of NO1, a New Sigma 2 Receptor/TMEM97 Fluorescent Ligand, Downregulates SOCE and Promotes Apoptosis in the Triple Negative Breast Cancer Cell Lines

Cancers

(1) Background: The structure of the Sigma 2 receptor/TMEM97 (σ2RTMEM97) has recently been report... more (1) Background: The structure of the Sigma 2 receptor/TMEM97 (σ2RTMEM97) has recently been reported. (2, 3) Methods and results: We used genetic and biochemical approaches to identify the molecular mechanism downstream of σ2R/TMEM97. The novel σ2R/TMEM97 fluorescent ligand, NO1, reduced the proliferation and survival of the triple negative breast cancer cell lines (TNBC: MDA-MB-231 and MDA-MB-468 cell lines), due to NO1-induced apoptosis. Greater bioaccumulation and faster uptake of NO1 in MDA-MB-231 cells compared to MCF10A or MCF7 cell lines were also shown. Accordingly, elevated σ2R/TMEM97 expression was confirmed by Western blotting. In contrast to NO1, other σ2R/TMEM97 ligands, such as SM21 and PB28, enhanced MDA-MB-231 cell proliferation and migration. Store-operated calcium entry (SOCE) is crucial for different cancer hallmarks. Here, we show that NO1, but not other σ2R/TMEM97 ligands, reduced SOCE in MDA-MB-231 cells. Similarly, TMEM97 silencing in MDA-MB-231 cells also impaired SOCE. NO1 administration downregulated STIM1-Orai1 interaction, probably by impairing the positive regulatory effect of σ2R/TMEM97 on STIM1, as we were unable to detect interaction with Orai1. (4) Conclusion: σ2R/TMEM97 is a key protein for the survival of triple negative breast cancer cells by promoting SOCE; therefore, NO1 may become a good pharmacological tool to avoid their proliferation.

Research paper thumbnail of TRPC Channels in the SOCE Scenario

Cells

Transient receptor potential (TRP) proteins form non-selective Ca2+ permeable channels that contr... more 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 participati...

Research paper thumbnail of TRPC channels: Dysregulation and Ca2+ Mishandling In Ischemic Heart Disease

Transient receptor potential canonical (TRPC) channels are ubiquitously expressed in excitable an... more 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, TRPC may form homo or heterotetrameric ion channel, 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 of the expression of different TRPC isoforms has been observed in different animal model of heart infarcts, and in vitro experimental model of ischemia and reperfusion. TRPC-mediated increase of the intracellular Ca2+ concentration seems required for the activation of 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 angi...

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

Cancers

Orai1 plays a major role in store-operated Ca2+ entry (SOCE) in triple-negative breast cancer (TN... more Orai1 plays a major role in store-operated Ca2+ 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 Ca2+ responses to agonists. The Ca2+ calmodulin-activated adenylyl cyclase type 8 (AC8) was reported to interact directly with Orai1, thus mediating a dynamic interplay between the Ca2+- 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 Ca2+ ent...

Research paper thumbnail of {"__content__"=>"Store-Operated Ca Entry in Breast Cancer Cells: Remodeling and Functional Role.", "sup"=>{"__content__"=>"2+"}}

International journal of molecular sciences, Jan 14, 2018

Breast cancer is the most common type of cancer in women. It is a heterogeneous disease that rang... more 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.

Research paper thumbnail of TRP Channels in Angiogenesis and Other Endothelial Functions

Frontiers in Physiology

Angiogenesis is the growth of blood vessels mediated by proliferation, migration, and spatial org... more Angiogenesis is the growth of blood vessels mediated by proliferation, migration, and spatial organization of endothelial cells. This mechanism is regulated by a balance between stimulatory and inhibitory factors. Proangiogenic factors include a variety of VEGF family members, while thrombospondin and endostatin, among others, have been reported as suppressors of angiogenesis. Transient receptor potential (TRP) channels belong to a superfamily of cation-permeable channels that play a relevant role in a number of cellular functions mostly derived from their influence in intracellular Ca 2+ homeostasis. Endothelial cells express a variety of TRP channels, including members of the TRPC, TRPV, TRPP, TRPA, and TRPM families, which play a relevant role in a number of functions, including endothelium-induced vasodilation, vascular permeability as well as sensing hemodynamic and chemical changes. Furthermore, TRP channels have been reported to play an important role in angiogenesis. This review summarizes the current knowledge and limitations concerning the involvement of particular TRP channels in growth factor-induced angiogenesis.

Research paper thumbnail of EFHB is a Novel Cytosolic Ca<sup>2+</sup> Sensor That Modulates STIM1-SARAF Interaction

Cellular Physiology and Biochemistry

Background/Aims: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Amon... more 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 SOC...

Research paper thumbnail of Filamin A modulates platelet function

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

Cancers, Jan 14, 2018

Transient receptor potential channels convey signaling information from a number of stimuli to a ... more 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. Fi...

Research paper thumbnail of Fine-tuning of microRNAs in type 2 diabetes mellitus

Current medicinal chemistry, Dec 5, 2017

Type 2 diabetes mellitus is a metabolic disease widely spread across industrialized countries. Se... more 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 miRNA involved in pancreas growth and development, insulin...

Research paper thumbnail of {"__content__"=>"Urocortin-2 Prevents Dysregulation of Ca Homeostasis and Improves Early Cardiac Remodeling After Ischemia and Reperfusion.", "sup"=>{"__content__"=>"2+"}}

Frontiers in physiology, 2018

Urocortin-2 (Ucn-2) is a potent cardioprotector against Ischemia and Reperfusion (I/R) injuries. ... more Urocortin-2 (Ucn-2) is a potent cardioprotector against Ischemia and Reperfusion (I/R) injuries. However, little is known about its role in the regulation of intracellular Ca concentration ([Ca]) under I/R. Here, we examined whether the addition of Ucn-2 in reperfusion promotes cardioprotection focusing on ([Ca] handling. Cardiac Wistar rat model of I/R was induced by transient ligation of the left coronary artery and experiments were conducted 1 week after surgery in tissue and adult cardiomyocytes isolated from risk and remote zones. We observed that I/R promoted significant alteration in cardiac contractility as well as an increase in hypertrophy and fibrosis in both zones. The study of confocal [Ca] imaging in adult cardiomyocytes revealed that I/R decreased the amplitude of [Ca] transient and cardiomyocytes contraction in risk and remote zones. Interestingly, intravenous infusion of Ucn-2 before heart's reperfusion recovered significantly cardiac contractility and prevented...

Research paper thumbnail of Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

Cellular and Molecular Life Sciences

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-acti... more 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 plas...

Research paper thumbnail of prodomain ppFurin enhances SOCE through TRPC 6 activation in 2 breast cancer cells 3 4

The intracellular calcium concentration ([Ca 2+ ]i) modulation plays a key role in the regulation... more The intracellular calcium concentration ([Ca 2+ ]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca 2+ ]i homeostasis is involved in tumor initiation and progression. Here we show that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced SOCE through TRPC6 activation that associated reduced cells malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments, revealed that the ppFurin-expressing cells are unable to process adequately the PC substrates proVEGF-C and proIGF-1R. Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 significantly induces Ca 2+ entry which is impaired by silencing of TRPC6 expression. Analysis of TRPC6 activation revealed its up-regulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. The expression of ppFurin in MDA-MB-231 cells reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca 2+ mobilization leading to breast cancer cells malignant phenotype repression and reduction of their resistance to treatments.

Research paper thumbnail of Protein and Vesicle Trafficking , Cytoskeleton Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca 2 entry in mouse pancreatic acinar cells

Rosado, Juan A., Pedro C. Redondo, Ginés M. Salido, Stewart O. Sage, and Jose A. Pariente. Cleava... more Rosado, Juan A., Pedro C. Redondo, Ginés M. Salido, Stewart O. Sage, and Jose A. Pariente. Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca entry in mouse pancreatic acinar cells. Am J Physiol Cell Physiol 288: C214–C221, 2005. First published September 8; doi:10.1152/ajpcell.00241.2004.—We recently reported that store-operated Ca entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as “secretion-like coupling.” As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosomeassociated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stim...

Research paper thumbnail of Role of Orai3 in the Pathophysiology of Cancer

International Journal of Molecular Sciences

The mammalian exclusive Orai3 channel participates in the generation and/or modulation of two ind... more 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 ass...

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

Biochemical Journal

TRPC6 forms non-selective cation channels activated by a variety of stimuli that are involved in ... more 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 cytosol...

Research paper thumbnail of Furin Prodomain ppFurin Enhances Ca2+ Entry Through Orai and TRPC6 Channels’ Activation in Breast Cancer Cells

Cancers

The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation o... more The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we showed that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced store-operated calcium entry (SOCE) and reduced the cell malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments revealed that the ppFurin-expressing cells are unable to adequately process the proprotein convertase (PC) substrates vascular endothelial growth factor C (proVEGF-C) and insulin-like growth factor-1 receptor (proIGF-1R). Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 and BT20 cells significantly enhanced SOCE...

Research paper thumbnail of PGRMC1 Inhibits Progesterone-Evoked Proliferation and Ca2+ Entry Via STIM2 in MDA-MB-231 Cells

International Journal of Molecular Sciences

Progesterone receptor membrane component 1 (PGRMC1) has been shown to regulate some cancer hallma... more 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...

Research paper thumbnail of Experiencias de Docencia Virtual en Facultades de Medicina Españolas durante la pandemia COVID-19 (I): Anatomía, Fisiología, Fisiopatología, Oncología

Revista Española de Educación Médica

Presentamos un resumen de las actividades que algunos profesores de Facultades de Medicina españo... more Presentamos un resumen de las actividades que algunos profesores de Facultades de Medicina españolas han llevado a cabo durante las 3 semanas previas a las vacaciones de primavera. En éstas, la docencia presencial tuvo que ser sustituída por actividades en línea o virtuales, a causa de la implantación del estado de alarma en España, que motivó el cierre completo de las Universidades desde el 13 de marzo de 2020. We present a summary of the activities that some professors of Schools of Medicine of Spain have carried out during the 3 weeks prior to spring break. In these, face-to-face teaching had to be replaced by online or virtual activities, due to the implementation of the state of alarm in Spain, which led to the complete closure of the Universities since March 13, 2020.

Research paper thumbnail of Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry

International Journal of Molecular Sciences

Arachidonic acid (AA) is a phospholipase A2 metabolite that has been reported to mediate a pletho... more 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 eff...

Research paper thumbnail of NO1, a New Sigma 2 Receptor/TMEM97 Fluorescent Ligand, Downregulates SOCE and Promotes Apoptosis in the Triple Negative Breast Cancer Cell Lines

Cancers

(1) Background: The structure of the Sigma 2 receptor/TMEM97 (σ2RTMEM97) has recently been report... more (1) Background: The structure of the Sigma 2 receptor/TMEM97 (σ2RTMEM97) has recently been reported. (2, 3) Methods and results: We used genetic and biochemical approaches to identify the molecular mechanism downstream of σ2R/TMEM97. The novel σ2R/TMEM97 fluorescent ligand, NO1, reduced the proliferation and survival of the triple negative breast cancer cell lines (TNBC: MDA-MB-231 and MDA-MB-468 cell lines), due to NO1-induced apoptosis. Greater bioaccumulation and faster uptake of NO1 in MDA-MB-231 cells compared to MCF10A or MCF7 cell lines were also shown. Accordingly, elevated σ2R/TMEM97 expression was confirmed by Western blotting. In contrast to NO1, other σ2R/TMEM97 ligands, such as SM21 and PB28, enhanced MDA-MB-231 cell proliferation and migration. Store-operated calcium entry (SOCE) is crucial for different cancer hallmarks. Here, we show that NO1, but not other σ2R/TMEM97 ligands, reduced SOCE in MDA-MB-231 cells. Similarly, TMEM97 silencing in MDA-MB-231 cells also impaired SOCE. NO1 administration downregulated STIM1-Orai1 interaction, probably by impairing the positive regulatory effect of σ2R/TMEM97 on STIM1, as we were unable to detect interaction with Orai1. (4) Conclusion: σ2R/TMEM97 is a key protein for the survival of triple negative breast cancer cells by promoting SOCE; therefore, NO1 may become a good pharmacological tool to avoid their proliferation.

Research paper thumbnail of TRPC Channels in the SOCE Scenario

Cells

Transient receptor potential (TRP) proteins form non-selective Ca2+ permeable channels that contr... more 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 participati...

Research paper thumbnail of TRPC channels: Dysregulation and Ca2+ Mishandling In Ischemic Heart Disease

Transient receptor potential canonical (TRPC) channels are ubiquitously expressed in excitable an... more 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, TRPC may form homo or heterotetrameric ion channel, 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 of the expression of different TRPC isoforms has been observed in different animal model of heart infarcts, and in vitro experimental model of ischemia and reperfusion. TRPC-mediated increase of the intracellular Ca2+ concentration seems required for the activation of 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 angi...

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

Cancers

Orai1 plays a major role in store-operated Ca2+ entry (SOCE) in triple-negative breast cancer (TN... more Orai1 plays a major role in store-operated Ca2+ 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 Ca2+ responses to agonists. The Ca2+ calmodulin-activated adenylyl cyclase type 8 (AC8) was reported to interact directly with Orai1, thus mediating a dynamic interplay between the Ca2+- 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 Ca2+ ent...

Research paper thumbnail of {"__content__"=>"Store-Operated Ca Entry in Breast Cancer Cells: Remodeling and Functional Role.", "sup"=>{"__content__"=>"2+"}}

International journal of molecular sciences, Jan 14, 2018

Breast cancer is the most common type of cancer in women. It is a heterogeneous disease that rang... more 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.

Research paper thumbnail of TRP Channels in Angiogenesis and Other Endothelial Functions

Frontiers in Physiology

Angiogenesis is the growth of blood vessels mediated by proliferation, migration, and spatial org... more Angiogenesis is the growth of blood vessels mediated by proliferation, migration, and spatial organization of endothelial cells. This mechanism is regulated by a balance between stimulatory and inhibitory factors. Proangiogenic factors include a variety of VEGF family members, while thrombospondin and endostatin, among others, have been reported as suppressors of angiogenesis. Transient receptor potential (TRP) channels belong to a superfamily of cation-permeable channels that play a relevant role in a number of cellular functions mostly derived from their influence in intracellular Ca 2+ homeostasis. Endothelial cells express a variety of TRP channels, including members of the TRPC, TRPV, TRPP, TRPA, and TRPM families, which play a relevant role in a number of functions, including endothelium-induced vasodilation, vascular permeability as well as sensing hemodynamic and chemical changes. Furthermore, TRP channels have been reported to play an important role in angiogenesis. This review summarizes the current knowledge and limitations concerning the involvement of particular TRP channels in growth factor-induced angiogenesis.

Research paper thumbnail of EFHB is a Novel Cytosolic Ca<sup>2+</sup> Sensor That Modulates STIM1-SARAF Interaction

Cellular Physiology and Biochemistry

Background/Aims: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Amon... more 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 SOC...

Research paper thumbnail of Filamin A modulates platelet function

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

Cancers, Jan 14, 2018

Transient receptor potential channels convey signaling information from a number of stimuli to a ... more 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. Fi...

Research paper thumbnail of Fine-tuning of microRNAs in type 2 diabetes mellitus

Current medicinal chemistry, Dec 5, 2017

Type 2 diabetes mellitus is a metabolic disease widely spread across industrialized countries. Se... more 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 miRNA involved in pancreas growth and development, insulin...

Research paper thumbnail of {"__content__"=>"Urocortin-2 Prevents Dysregulation of Ca Homeostasis and Improves Early Cardiac Remodeling After Ischemia and Reperfusion.", "sup"=>{"__content__"=>"2+"}}

Frontiers in physiology, 2018

Urocortin-2 (Ucn-2) is a potent cardioprotector against Ischemia and Reperfusion (I/R) injuries. ... more Urocortin-2 (Ucn-2) is a potent cardioprotector against Ischemia and Reperfusion (I/R) injuries. However, little is known about its role in the regulation of intracellular Ca concentration ([Ca]) under I/R. Here, we examined whether the addition of Ucn-2 in reperfusion promotes cardioprotection focusing on ([Ca] handling. Cardiac Wistar rat model of I/R was induced by transient ligation of the left coronary artery and experiments were conducted 1 week after surgery in tissue and adult cardiomyocytes isolated from risk and remote zones. We observed that I/R promoted significant alteration in cardiac contractility as well as an increase in hypertrophy and fibrosis in both zones. The study of confocal [Ca] imaging in adult cardiomyocytes revealed that I/R decreased the amplitude of [Ca] transient and cardiomyocytes contraction in risk and remote zones. Interestingly, intravenous infusion of Ucn-2 before heart's reperfusion recovered significantly cardiac contractility and prevented...