STIM1 translocation to the nucleus protects cells from DNA damage.

DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at studying the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as to the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, which explains the increased sensitivity of STIM1-KO cells to MMC. This study not only unveils a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expands our understanding of the genes involved in DNA repair.

Internalized Amyloid-ß (1-42) Peptide Inhibits the Store-Operated Calcium Entry in HT-22 Cells.

Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer's disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-ß peptides (Aß) in AD. In recent works, we have proposed that Aß-calmodulin (CaM) complexation may play a major role in neuronal Ca2+ signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 µM Aß(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aß(1-42), respectively. Internalized Aß(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aß. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aß(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aß(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aß(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca2+ homeostasis can be detected, and is elicited by only 2 h of incubation with 2 µM Aß(1-42) oligomers. We conclude that Aß(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca2+-leak channels.

Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation.

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621 Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621 These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.

STIM1 Deficiency Leads to Specific Down-Regulation of ITPR3 in SH-SY5Y Cells.

STIM1 is an endoplasmic reticulum (ER) protein that modulates the activity of a number of Ca2+ transport systems. By direct physical interaction with ORAI1, a plasma membrane Ca2+ channel, STIM1 activates the ICRAC current, whereas the binding with the voltage-operated Ca2+ channel CaV1.2 inhibits the current through this latter channel. In this way, STIM1 is a key regulator of Ca2+ signaling in excitable and non-excitable cells, and altered STIM1 levels have been reported to underlie several pathologies, including immunodeficiency, neurodegenerative diseases, and cancer. In both sporadic and familial Alzheimer's disease, a decrease of STIM1 protein levels accounts for the alteration of Ca2+ handling that compromises neuronal cell viability. Using SH-SY5Y cells edited by CRISPR/Cas9 to knockout STIM1 gene expression, this work evaluated the molecular mechanisms underlying the cell death triggered by the deficiency of STIM1, demonstrating that STIM1 is a positive regulator of ITPR3 gene expression. ITPR3 (or IP3R3) is a Ca2+ channel enriched at ER-mitochondria contact sites where it provides Ca2+ for transport into the mitochondria. Thus, STIM1 deficiency leads to a strong reduction of ITPR3 transcript and ITPR3 protein levels, a consequent decrease of the mitochondria free Ca2+ concentration ([Ca2+]mit), reduction of mitochondrial oxygen consumption rate, and decrease in ATP synthesis rate. All these values were normalized by ectopic expression of ITPR3 in STIM1-KO cells, providing strong evidence for a new mode of regulation of [Ca2+]mit mediated by the STIM1-ITPR3 axis.

STIM1 phosphorylation triggered by epidermal growth factor mediates cell migration.

STIM1 is a key regulator of store-operated calcium entry (SOCE), and therefore a mediator of Ca²âº entry-dependent cellular events. Phosphorylation of STIM1 at ERK1/2 target sites has been described as enhancing STIM1 activation during intracellular Ca²âº emptying triggered by the inhibition of the sarco(endo)plasmic Ca²âº -ATPase with thapsigargin. However, no physiological function is known for this specific phosphorylation. The present study examined the role of STIM1 phosphorylation in cell signaling triggered by EGF. Using a human endometrial adenocarcinoma cell line (Ishikawa cells) EGF or H-Ras(G12V), an active mutant of H-Ras, was found to trigger STIM1 phosphorylation at residues Ser575, Ser608, and Ser621, and this process was sensitive to PD0325901, an inhibitor of ERK1/2. Both, ERK1/2 activation and STIM1 phosphorylation took place in the absence of extracellular Ca²âº, indicating that both events are upstream steps for Ca²âºentry activation. Also, EGF triggered the dissociation of STIM1 from EB1 (a regulator of microtubule plus-ends) in a manner similar to that reported for the activation of STIM1 by thapsigargin. Migration of the Ishikawa cells was impaired when STIM1 phosphorylation was targeted by Ser-to-Ala substitution mutation of ERK1/2 target sites. This effect was also observed with the Ca²âº channel blocker SKF96365. Phosphomimetic mutation of STIM1 restored the migration to levels similar to that found for STIM1-wild type. Finally, the increased vimentin expression and relocalization of E-cadherin triggered by EGF were largely inhibited by targeting STIM1 phosphorylation, while STIM1-S575E/S608E/S621E normalized the profiles of these two EMT markers.

Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.

STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE). Upon depletion of Ca(2+) concentration within the endoplasmic reticulum (ER), STIM1 relocalizes at ER-plasma membrane junctions, activating store-operated calcium channels (SOCs). Although the molecular details for STIM1-SOC binding is known, the regulation of SOCE remains largely unknown. A detailed list of phosphorylated residues within the STIM1 sequence has been reported. However, the molecular pathways controlling this phosphorylation and its function are still under study. Using phosphospecific antibodies, we demonstrate that ERK1/2 mediates STIM1 phosphorylation at Ser575, Ser608 and Ser621 during Ca(2+) store depletion, and that Ca(2+) entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. Although Ser to Ala mutation of residues 575, 608 and 621 showed a constitutive binding to EB1 even after Ca(2+) store depletion, Ser to Glu mutation of these residues (to mimic the phosphorylation profile attained after store depletion) triggered full dissociation from EB1. Given that wild-type STIM1 and STIM1(S575E/S608E/S621E) activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca(2+) store depletion, an event that is fully reversed by Ca(2+) entry and store refilling.

Intracellular cyclophilin A is an important Ca(2+) regulator in platelets and critically involved in arterial thrombus formation.

Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca(2+) activity ([Ca(2+)](i)). Activation-dependent depletion of intracellular Ca(2+) stores triggers Ca(2+) entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca(2+) sensor that regulates store-operated Ca(2+) entry through activation of the pore-forming subunit Orai1, the major store-operated Ca(2+) entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca(2+) modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca(2+) mobilization from intracellular stores and Ca(2+) influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca(2+) sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPA-deficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca(2+) regulator in platelets, a critical mechanism for arterial thrombosis.

Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry.

Store-operated calcium entry (SOCE) is an important Ca2+ entry pathway that regulates many cell functions. Upon store depletion, STIM1, a transmembrane protein located in the endoplasmic reticulum (ER), aggregates and relocates close to the plasma membrane (PM) where it activates store-operated calcium channels (SOCs). Although STIM1 was early defined as a phosphoprotein, the contribution of the phosphorylation has been elusive. In the present work, STIM1 was found to be a target of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in vitro, and we have defined the ERK1/2-phosphorylated sites on the STIM1 sequence. Using HEK293 cells stably transfected for the expression of tagged STIM1, we found that alanine substitution mutants of ERK1/2 target sites reduced SOCE significantly, suggesting that phosphorylation of these residues are required to fully accomplish SOCE. Indeed, the ERK1/2 inhibitors PD184352 and PD0325901 decreased SOCE in transfected cells. Conversely, 12-O-tetradecanoylphorbol-13-acetate, which activates ERK1/2, enhanced SOCE in cells expressing wild-type tagged STIM1, but did not potentiate Ca2+ influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca2+ influx without disturbing the aggregation of STIM1 upon store depletion and without affecting the relocalization in ER-PM punctae. However, our results suggest that STIM1 phosphorylation at ERK1/2 target sites can modulate SOCE by altering STIM1 binding to SOCs, because a significant decrease in FRET efficiency was observed between alanine substitution mutants of STIM1-GFP and ORAI1-CFP.

Calcium signaling in mouse oocyte maturation: the roles of STIM1, ORAI1 and SOCE.

Calcium handling is critical for the oocyte function, since the first steps of fertilization are dependent on the appropriate Ca(2+) mobilization to originate transient spikes of the cytosolic Ca(2+) concentration. It is well known that the Ca(2+) influx from the extracellular milieu is required to maintain this signaling in mammalian oocytes. However, the regulation of the Ca(2+) channels involved in this process is still unknown in oocytes. STIM1, a key regulator of store-operated Ca(2+) entry (SOCE), relocates in the mouse oocyte shortly after sperm stimulation, suggesting that SOCE is involved in the maintenance of cytosolic Ca(2+)-spiking in the fertilized oocyte. Here, we show that there is an up-regulation of the expression of STIM1 at the germinal vesicle breakdown stage, and this expression remains steady during following maturation stages. We found that oocytes express ORAI1, a store-operated Ca(2+) channel, and that ORAI1 expression level was stable during oocyte maturation. Immature oocytes showed no Ca(2+) entry and no increase in STIM1-ORAI1 colocalization in response to the store depletion induced by thapsigargin. On the contrary, in mature oocytes, STIM1-ORAI1 colocalization is enhanced 3-fold by depletion of Ca(2+) stores, enabling the activation of store-operated calcium channels and therefore Ca(2+) entry. Finally, the correlation between SOCE activation during the maturation of oocytes and STIM1-ORAI1 colocalization strongly suggests that ORAI1 is involved in the Ca(2+) entry pathway in the mature oocyte. SOCE up-regulation in the final stage of maturation is further evidence of a major role for SOCE in fully mature oocytes, and therefore in Ca(2+) signaling at fertilization.

RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence.

Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 and increasing the plasma membrane ruffling. The role of ORAI1 at the leading edge was studied in genetically engineered U2OS cells lacking ORAI1 expression that helped us to prove the key role of this Ca2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.

Relocalization of STIM1 in mouse oocytes at fertilization: early involvement of store-operated calcium entry.

Calcium waves represent one of the most important intracellular signaling events in oocytes at fertilization required for the exit from metaphase arrest and the resumption of the cell cycle. The molecular mechanism ruling this signaling has been described in terms of the contribution of intracellular calcium stores to calcium spikes. In this work, we considered the possible contribution of store-operated calcium entry (SOCE) to this signaling, by studying the localization of the protein STIM1 in oocytes. STIM1 has been suggested to play a key role in the recruitment and activation of plasma membrane calcium channels, and we show here that mature mouse oocytes express this protein distributed in discrete clusters throughout their periphery in resting cells, colocalizing with the endoplasmic reticulum marker calreticulin. However, immunolocalization of the endogenous STIM1 showed considerable redistribution over larger areas or patches covering the entire periphery of the oocyte during Ca(2+) store depletion induced with thapsigargin or ionomycin. Furthermore, pharmacological activation of endogenous phospholipase C induced a similar pattern of redistribution of STIM1 in the oocyte. Finally, fertilization of mouse oocytes revealed a significant and rapid relocalization of STIM1, similar to that found after pharmacological Ca(2+) store depletion. This particular relocalization supports a role for STIM1 and SOCE in the calcium signaling during early stages of fertilization.

Correction to: STIM1 deficiency is linked to Alzheimer's disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry.

The original publication of this paper contains errors.

Hydrogen sulfide raises cytosolic calcium in neurons through activation of L-type Ca2+ channels.

Hydrogen sulfide (H(2)S) concentration can be maintained in cell cultures within the range reported for rat brain by repetitive pulses of sodium hydrogen sulfide. Less than 2 h exposure to H(2)S concentrations within 50 and 120 microM (i.e., within the upper segment of the reported physiological range of H(2)S in rat brain), produces a large shift of the intracellular calcium homeostasis in cerebellar granule neurons (CGN) in culture, leading to a large and sustained increase of cytosolic calcium concentration. Only 1 h exposure to H(2)S concentrations within 100 and 300 microM raises intracellular calcium to the neurotoxic range, with nearly 50% cell death after 2 h. L-type Ca(2+) channels antagonists nimodipine and nifedipine block both the H(2)S-induced rise of cytosolic calcium and cell death. The N-methyl-D-aspartate receptor antagonists (+)-MK-801 and DL-2-amino-5-phosphonovaleric acid afforded a nearly complete protection against H(2)S-induced CGN death and largely attenuated the rise of cytosolic calcium. Thus, H(2)S-induced rise of cytosolic calcium eventually reaches the neurotoxic cytosolic calcium range, leading to glutamate-induced excitotoxic CGN death. The authors conclude that H(2)S is a major modulator of calcium homeostasis in neurons as it induces activation of Ca(2+) entry through L-type Ca(2+) channels, and thereby of neuronal activity.

Reduction of ascorbate free radical by the plasma membrane of synaptic terminals from rat brain.

Synaptic plasma membranes (SPMV) decrease the steady state ascorbate free radical (AFR) concentration of 1mM ascorbate in phosphate/EDTA buffer (pH 7), due to AFR recycling by redox coupling between ascorbate and the ubiquinone content of these membranes. In the presence of NADH, but not NADPH, SPMV catalyse a rapid recycling of AFR which further lower the AFR concentration below 0.05 microM. These results correlate with the nearly 10-fold higher NADH oxidase over NADPH oxidase activity of SPMV. SPMV has NADH-dependent coenzyme Q reductase activity. In the presence of ascorbate the stimulation of the NADH oxidase activity of SPMV by coenzyme Q(1) and cytochrome c can be accounted for by the increase of the AFR concentration generated by the redox pairs ascorbate/coenzyme Q(1) and ascorbate/cytochrome c. The NADH:AFR reductase activity makes a major contribution to the NADH oxidase activity of SPMV and decreases the steady-state AFR concentration well below the micromolar concentration range.

Contribution of culture media to oxidative stress and its effect on human oocytes.

The adverse effects of reactive oxygen species (ROS) on many aspects of reproduction are well documented. However, much less is known regarding the contribution of culture media to the oxidative stress of gametes during assisted reproductive techniques. This study measured the generation of ROS by culture media during IVF procedures and its effects on human oocytes. Commercially supplied culture media generated ROS at various rates, depending on the composition, whereas follicular fluid generated ROS at a much lower level. The incubation of cumulus-oocyte complexes (COC) in culture media induced marked lipid peroxidation compared with levels found in freshly retrieved COC. This plasma membrane damage, measured with the quenching of cis-parinaric acid fluorescence assay, was attenuated by supplementation of the medium with alpha-tocopherol or catalase. Moreover, there was an association between ROS production by culture medium and thiolic content consumption within the oocytes, suggesting that the intracellular reduced glutathione pool was partially depleted during in-vitro manipulation. The results show that culture medium could damage oocytes (and consequently embryo development) depending on their composition, and it is proposed that current IVF protocols could be revised in order to decrease ROS generation.

Role of STIM1 in neurodegeneration.

STIM1 is an endoplasmic reticulum (ER) protein with a key role in Ca2+ mobilization. Due to its ability to act as an ER-intraluminal Ca2+ sensor, it regulates store-operated Ca2+ entry (SOCE), which is a Ca2+ influx pathway involved in a wide variety of signalling pathways in eukaryotic cells. Despite its important role in Ca2+ transport, current knowledge about the role of STIM1 in neurons is much more limited. Growing evidence supports a role for STIM1 and SOCE in the preservation of dendritic spines required for long-term potentiation and the formation of memory. In this regard, recent studies have demonstrated that the loss of STIM1, which impairs Ca2+ mobilization in neurons, risks cell viability and could be the cause of neurodegenerative diseases. The role of STIM1 in neurodegeneration and the molecular basis of cell death triggered by low levels of STIM1 are discussed in this review.

STIM1 deficiency is linked to Alzheimer's disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry.

STIM1 is an endoplasmic reticulum protein with a role in Ca2+ mobilization and signaling. As a sensor of intraluminal Ca2+ levels, STIM1 modulates plasma membrane Ca2+ channels to regulate Ca2+ entry. In neuroblastoma SH-SY5Y cells and in familial Alzheimer's disease patient skin fibroblasts, STIM1 is cleaved at the transmembrane domain by the presenilin-1-associated γ-secretase, leading to dysregulation of Ca2+ homeostasis. In this report, we investigated expression levels of STIM1 in brain tissues (medium frontal gyrus) of pathologically confirmed Alzheimer's disease patients, and observed that STIM1 protein expression level decreased with the progression of neurodegeneration. To study the role of STIM1 in neurodegeneration, a strategy was designed to knock-out the expression of STIM1 gene in the SH-SY5Y neuroblastoma cell line by CRISPR/Cas9-mediated genome editing, as an in vitro model to examine the phenotype of STIM1-deficient neuronal cells. It was proved that, while STIM1 is not required for the differentiation of SH-SY5Y cells, it is absolutely essential for cell survival in differentiating cells. Differentiated STIM1-KO cells showed a significant decrease of mitochondrial respiratory chain complex I activity, mitochondrial inner membrane depolarization, reduced mitochondrial free Ca2+ concentration, and higher levels of senescence as compared with wild-type cells. In parallel, STIM1-KO cells showed a potentiated Ca2+ entry in response to depolarization, which was sensitive to nifedipine, pointing to L-type voltage-operated Ca2+ channels as mediators of the upregulated Ca2+ entry. The stable knocking-down of CACNA1C transcripts restored mitochondrial function, increased mitochondrial Ca2+ levels, and dropped senescence to basal levels, demonstrating the essential role of the upregulation of voltage-operated Ca2+ entry through Cav1.2 channels in STIM1-deficient SH-SY5Y cell death. KEY MESSAGES: STIM1 protein expression decreases with the progression of neurodegeneration in Alzheimer's disease. STIM1 is essential for cell viability in differentiated SH-SY5Y cells. STIM1 deficiency triggers voltage-regulated Ca2+ entry-dependent cell death. Mitochondrial dysfunction and senescence are features of STIM1-deficient differentiated cells.

Blood micromolar concentrations of kaempferol afford protection against ischemia/reperfusion-induced damage in rat brain.

The slow time course of neurodegeneration after brain ischemia/reperfusion opened a realistic time window for the application of protective therapies to prevent spreading of brain damage. In this work, we studied the ability of micromolar concentrations of this flavonoid in the blood to protect against brain damage induced by transient focal cerebral ischemia in rats. Transient focal cerebral ischemia was induced by middle cerebral artery occlusion in adult rats and brain damage has been monitored by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H-E) staining, 'in situ' terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL), 'in situ' metalloproteinase activity using DQ-gelatin and loss of anti-laminin staining. Intravenous injections of kaempferol, at a dose of 10-15 mumol/L of blood 30 min before the induction of a 60 min ischemia-episode and just after reperfusion, led to >90% and 70-80% (TTC, H-E, TUNEL) decrease of brain damage in the temporal-frontal areas of neocortex and striatum, respectively, but only 40-50% decrease of brain damage was observed in the hippocampus and vicinal caudal areas of the striatum. This treatment with kaempferol also produced a similar reduction of metalloproteinase activation and loss of anti-laminin staining in cortical and striatum infarct areas. Kaempferol treatment efficiently protected against nitrosative-oxidative stress after ischemia/reperfusion, as shown by nearly complete protection against the increase of protein nitrotyrosines, and also afforded strong protection against the increase of apoptotic cell death (TUNEL) and biochemical markers of apoptosis, such as caspase-9 activity and poly-(ADP-ribose) polymerase degradation. On these grounds, a potential new therapeutic role of kaempferol to acute treatment of ischemic stroke is suggested.

Store-operated calcium entry is dispensable for the activation of ERK1/2 pathway in prostate cancer cells.

STIM1, the endoplasmic reticulum Ca2+ sensor that modulates the activity of plasma membrane Ca2+ channels, becomes phosphorylated at ERK1/2 target sites during Ca2+ store depletion triggered by thapsigargin or epidermal growth factor (EGF). This ERK1/2-dependent phosphorylation regulates STIM1 localization and dissociation from microtubules, and it is known that enhances the binding to ORAI1, a store-operated Ca2+ entry (SOCE) channel, leading to the activation of this Ca2+ influx pathway. However, there remained some evidence of a role for SOCE in the activation of ERK1/2, and here we assessed the contribution of SOCE to ERK1/2 activation by generating a STIM1-deficient cell line by CRISPR/Cas9 genome editing of the STIM1 locus in prostate cancer PC3 cells. The genomic modification consisted of a 16 base-pair insertion in exon 5 of both alleles, therefore abrogating STIM1 synthesis. STIM1-KO cells did show a striking decrease in Ca2+ influx in response to thapsigargin or EGF, a result that demonstrates that SOCE mediates Ca2+ entry in PC3 cells during stimulation with EGF. Moreover, identical levels of total ERK1/2 were found in STIM1-KO cells and the parental cell line, and ERK1/2 activation was fully activated in KO cells, both in the presence and in the absence of extracellular Ca2+, a result that supports that STIM1 and SOCE are not required for ERK1/2 activation. This activation was sensitive to Src kinase inhibition, but not to CAMKII nor PKC inhibition, a result that sets STIM1 and SOCE as downstream targets of the axis Src-Raf-MEK-ERK, rather than upstream regulators.