Francisco Javier Martin-Romero

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.

Fluorescence measurements of steady state peroxynitrite production upon SIN-1 decomposition: NADH versus dihydrodichlorofluorescein and dihydrorhodamine 123.

The production of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition can be continuously monitored, with a sensitivity ≤ 0.1 μM, from the kinetics of NADH fluorescence quenching in phosphate buffers, as well as in buffers commonly used with cell cultures, like Lockes buffer or Dulbeccos modified Eagles medium (DMEM-F12). The half-time for peroxynitrite production during SIN-1 decomposition ranged from 14–18 min in DMEM-F12 (plus and minus phenol red) to 21.5 min in Lockes buffer and 26 min in DMEM-F12 supplemented with apotransferrin (0.1 mg/mL). The concentration of peroxynitrite reached a peak that was linearly dependent upon SIN-1 concentration, and that for 100 μM SIN-1 amounted to 1.4 ± 0.2 μM in Lockes buffer, 3.2–3.6 μM in DMEM-F12 (plus and minus phenol red) and 1.8 μM in DMEM-F12 supplemented with apotransferrin. Thus, the maximum concentration of peroxynitrite ranged from 1.2 to 3.6% of added SIN-1. NADH was found to be less sensitive than dihydrorhodamine 123 and 2′,7′-dichlorodihydrofluorescein diacetate to oxidation by H2O2, which is produced during SIN-1 decomposition in common buffers. It is shown that peroxynitrite concentration can be controlled (±5%) during predetermined times by using sequential SIN-1 pulses, to simulate chronic exposure of cells or subcellular components to peroxynitrite.

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.

Inhibition of oxidative stress produced by plasma membrane NADH oxidase delays low-potassium-induced apoptosis of cerebellar granule cells.

From 1 to 3 h after the onset of cerebellar granule cells (CGC) apoptosis in a low‐K+(5 mm KCl) medium there was a large decay of NADH and a 2.5‐fold increase of the rate of reactive oxygen species (ROS) production (measured using CGC loaded with dichlorodihydrofluorescein). During the same time period, the ascorbate‐dependent NADH oxidase activity, which accounted for more than 90% of both total NADH oxidase activity and NADH‐dependent ·O2– production of CGC lysates, increased 2.5‐ to threefold. The stimulation of the ascorbate‐dependent NADH oxidase activity by oxidized cytochrome c, 2.5‐fold at saturation with a K0.5 of 4–5 µm cytochrome c, can at least partially explain this activation. The plasma membrane ascorbate‐dependent NADH oxidase activity accounted for more than 70% of the total activity (both in terms of NADH oxidase and ·O2– release) of CGC lysates. 4‐Hydroxyquinazoline (4‐HQ), which was found to block this apoptotic process, prevented the increase of ROS production. 4‐HQ protection against cell viability loss and DNA fragmentation correlated with the inhibition by 4‐HQ of the ascorbate‐dependent NADH oxidase activity of CGC lysates, showing the same K0.5‐value (4–5 mm 4‐HQ). The efficient blockade of CGC apoptosis by addition of superoxide dismutase to the medium further supports the neurotoxic role of ·O2– overproduction by the plasma membrane ascorbate‐dependent NADH oxidase.

Intracellular cyclophilin A is an important Ca2+ 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.

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/ nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES‐containing Lockes buffer (pH 7.4) to 50–100 µm SIN‐1 during 2 h decreased the steady‐state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 ± 33 nm to 60 ± 10 nm, whereas exposure to ≥ 0.3 mm SIN‐1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 ± 10 nm (due to inactivation of L‐type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN‐1‐induced necrotic cell death. Both effects of SIN‐1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4‐benzoic acid)porphyrin chloride. Supplementation of Lockes buffer with catalase before addition of 0.5–1 mm SIN‐1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60–120 min exposure to SIN‐1 and also protected against SIN‐1‐induced necrotic cell death. α‐Tocopherol, the potent NMDA receptor antagonist (+)‐MK‐801 and the N‐ and P‐type Ca2+ channels blocker ω‐conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN‐1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60–120 min exposure to 0.5–1 mm SIN‐1. It is concluded that L‐type Ca2+ channels are a primary target of SIN‐1‐induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5–1 μm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.

Potassium-induced apoptosis in rat cerebellar granule cells involves cell-cycle blockade at the G1/S transition.

The role of regulators controlling the G1/S transition of the cell cycle was analyzed during neuronal apoptosis in post-mitotic cerebellar granule cells in an attempt to identify common mechanisms of control with transformed cells. Cyclin D1 and its associated kinase activity CDK4 (cyclin-dependent kinase 4) are major regulators of the G1/S transition. Whereas cyclin D1 is the regulatory subunit of the complex, CDK4 represents the catalytic domain that, once activated, will phosphorylate downstream targets such as the retinoblastoma protein, allowing cell-cycle progression. Apoptosis was induced in rat cerebellar granule cells by depleting potassium in presence of serum. Western-blot analyses were performed and protein kinase activities were measured. As apoptosis proceeded, loss in cell viability was coincident with a significant increase in cyclin D1 protein levels, whereas CDK4 expression remained essentially constant. Synchronized to cyclin D1 accumulation, cyclin-dependent kinase inhibitor p27Kip1 drastically dropped to 20% normal values. Cyclin D1/CDK4-dependent kinase activity increased early during apoptosis, reaching a maximum at 9–12 h and decreasing to very low levels by 48 h. Cyclin E, a major downstream target of cyclin D1, decreased concomitantly to the reduction in cyclin D1/CDK4-dependent kinase activity. We suggest that neuronal apoptosis takes place through functional alteration of proteins involved in the control of the G1/S transition of the cell cycle. Thus, apoptosis in post-mitotic neurons could result from a failed attempt to re-enter cell cycle in response to extracellular conditions affecting cell viability and it could involve mechanisms similar to those that promote proliferation in transformed cells.

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.

Store-Operated Calcium Entry in Human Oocytes and Sensitivity to Oxidative Stress

Abstract Calcium signaling is a cellular event that plays a key role at many steps of fertilization and early development. However, little is known regarding the contribution of extracellular Ca2+ influx into the cell to this signaling in gametes and early embryos. To better know the significance of calcium entry on oocyte physiology, we have evaluated the mechanism of store-operated calcium entry (SOCE) in human metaphase II (MII) oocytes and its sensitivity to oxidative stress, one of the major factors implicated in the outcome of in vitro fertilization (IVF) techniques. We show that depletion of intracellular Ca2+ stores through inhibition of sarco(endo)plasmic Ca2+-ATPase with thapsigargin triggers Ca2+ entry in resting human oocytes. Ba2+ and Mn2+ influx was also stimulated following inhibition, and Ca2+ entry was sensitive to pharmacological inhibition because the SOCE blocker 2-aminoethoxydiphenylborate (2-APB) reduced calcium and barium entry. These results support the conclusion that there is a plasma membrane mechanism responsible for the capacitative divalent cation entry in human oocytes. Moreover, the Ca2+ entry mechanism described in MII oocytes was found to be highly sensitive to oxidative stress. Hydrogen peroxide, at micromolar concentrations that could mimic culture conditions in IVF, elicited an increase of [Ca2+]i that was dependent on the presence of extracellular Ca2+. This rise was preventable by 2-APB, indicating that it was mainly due to the enhanced influx through store-operated calcium channels. In sum, our results demonstrate the occurrence of SOCE in human MII oocytes and the modification of this pathway due to oxidative stress, with possible consequences in IVF.

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

Summary STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE). Upon depletion of Ca2+ 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 Ca2+ store depletion, and that Ca2+ 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 Ca2+ 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 STIM1S575E/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 Ca2+ store depletion, an event that is fully reversed by Ca2+ entry and store refilling.