Joan-Marc Servitja

Regulation of p73 by c-Abl through the p38 MAP kinase pathway

p73 is a novel member of the p53 family of tumor suppressor proteins which is involved in cellular differentiation, tumor suppression, and the response to genotoxic stress. The molecular mechanisms regulating p73 activity are still poorly understood. Recently, p73 was found to be a target of the enzymatic activity of c-Abl, a non-receptor tyrosine kinase that potently activated in response to DNA damage. Here, we present evidence that c-Abl induces the phosphorylation of p73 in threonine residues adjacent to prolines, and that the p38 MAP kinase pathway mediates this response. Furthermore, we found that activation of p38 is sufficient to enhance the stability of p73, and that the transcriptional activation of p73 by c-Abl requires the activity of p38. These findings indicate that members of the MAP kinases superfamily of signaling molecules can regulate p73, and support a role for the p38 MAP kinase in a novel biochemical pathway by which c-Abl regulates this p53-related molecule.

Effects of Oxidative Stress on Phospholipid Signaling in Rat Cultured Astrocytes and Brain Slices

Although reactive oxygen species (ROS) are conventionally viewed as toxic by‐products of cellular metabolism, a growing body of evidence suggests that they may act as signaling molecules. We have studied the effects of hydrogen peroxide (H2O2)‐induced oxidative stress on phospholipid signaling in cultured rat cortical astrocytes. H2O2 stimulated the formation of phosphatidic acid and the accumulation of phosphatidylbutanol, a product of the phospholipase D (PLD)‐catalyzed transphosphatidylation reaction. The effect of exogenous H2O2 on the PLD response was mimicked by menadione‐induced production of endogenous H2O2. Oxidative stress also elicited inositol phosphate accumulation resulting from phosphoinositide phospholipase C (PLC) activation. The PLD response to H2O2 was totally suppressed by chelation of both extracellular and cytosolic Ca2+ with EGTA and BAPTA/AM, respectively. Furthermore, H2O2‐induced PLD stimulation was completely abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide and chelerythrine and by PKC down‐regulation. Activation of PLD by H2O2 was also inhibited by the protein‐tyrosine kinase inhibitor genistein. Finally, H2O2 also stimulated both PLC and PLD in rat brain cortical slices. These results show for the first time that oxidative stress elicits phospholipid breakdown by both PLC and PLD in rat cultured astrocytes and brain slices.

Group I metabotropic glutamate receptors mediate phospholipase D stimulation in rat cultured astrocytes.

Abstract: We have studied the activation of phospholipase D (PLD) by glutamate in rat cultured astrocytes by measuring the PLD‐catalyzed formation of [32P]phosphatidylbutanol in [32P]Pi‐prelabeled cells, stimulated in the presence of butanol. Glutamate elicited the activation of PLD in cortical astrocytes but not in cortical neurons, whereas similar glutamate activation of phosphoinositide phospholipase C was found in both astrocytes and neurons. The extent of PLD stimulation by glutamate was similar in astrocytes from brain cortex and hippocampus, but no effect was found in cerebellar astrocytes. In cortical astrocytes, the glutamate response was insensitive to antagonists of ionotropic glutamate receptors and was reproduced by agonists of metabotropic glutamate receptors (mGluRs) with a rank order of agonist potency similar to that reported for group I mGluR‐mediated phosphoinositide phospholipase activation [quisqualate > (S)‐3,5‐dihydroxyphenylglycine > (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid]. The response to (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid was inhibited by the mGluR antagonist (S‐α‐methyl‐4‐carboxyphenylglycine and, less potently, by 1‐aminoindan‐1,5‐dicarboxylic acid and 4‐carboxyphenylglycine, two antagonists of group I mGluRs that display higher potency on mGluR1 than on mGluR5. The mGluR5‐selective agonist (RS)‐2‐chloro‐5‐hydroxyphenylglycine also activated PLD in astrocytes. These findings indicate the involvement of group I mGluRs, most likely mGluR5, in the glutamate activation of PLD in cultured rat cortical astrocytes.

Nurr1 Protein Is Required for N-Methyl-d-aspartic Acid (NMDA) Receptor-mediated Neuronal Survival

Background: The mechanism involved in activity-dependent survival of neurons in the central nervous system is not fully understood. Results: Nurr1 is involved in excitatory transmission-dependent survival of glutamatergic neurons by acting downstream CREB and upstream of BDNF. Conclusion: Nurr1 activation mediates activity-dependent survival of glutamatergic neurons. Significance: A novel function of Nurr1 in activity-dependent survival of glutamatergic neurons is reported. NMDA receptor (NMDAR) stimulation promotes neuronal survival during brain development. Cerebellar granule cells (CGCs) need NMDAR stimulation to survive and develop. These neurons differentiate and mature during its migration from the external granular layer to the internal granular layer, and lack of excitatory inputs triggers their apoptotic death. It is possible to mimic this process in vitro by culturing CGCs in low KCl concentrations (5 mm) in the presence or absence of NMDA. Using this experimental approach, we have obtained whole genome expression profiles after 3 and 8 h of NMDA addition to identify genes involved in NMDA-mediated survival of CGCs. One of the identified genes was Nurr1, a member of the orphan nuclear receptor subfamily Nr4a. Our results report a direct regulation of Nurr1 by CREB after NMDAR stimulation. ChIP assay confirmed CREB binding to Nurr1 promoter, whereas CREB shRNA blocked NMDA-mediated increase in Nurr1 expression. Moreover, we show that Nurr1 is important for NMDAR survival effect. We show that Nurr1 binds to Bdnf promoter IV and that silencing Nurr1 by shRNA leads to a decrease in brain-derived neurotrophic factor (BDNF) protein levels and a reduction of NMDA neuroprotective effect. Also, we report that Nurr1 and BDNF show a similar expression pattern during postnatal cerebellar development. Thus, we conclude that Nurr1 is a downstream target of CREB and that it is responsible for the NMDA-mediated increase in BDNF, which is necessary for the NMDA-mediated prosurvival effect on neurons.

Characterization of the metabotropic glutamate receptors mediating phospholipase C activation and calcium release in cerebellar granule cells: calcium-dependence of the phospholipase C response

In this study we have determined the metabotropic glutamate receptors (mGluRs) involved in the glutamate activation of phospholipase C (PLC) and Ca2+ mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca2+ modulation of the PLC response. Both PLC activation and Ca2+ signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1α and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca2+ for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re‐exposure of cells to Ca2+‐containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca2+ concentration. On the other hand, loading of the intracellular Ca2+ stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca2+ in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca2+ during stimulation and the chelation of cytosolic Ca2+ with BAPTA/AM inhibited both glutamate‐elicited Ca2+ response and PLC activation. Overall, these results indicate that the mGluR‐mediated activation of PLC depends on the presence of extracellular Ca2+ and can be modulated by moderate changes of cytosolic Ca2+. Furthermore, ryanodine reduced PLC stimulation by glutamate in predepolarized cells but not in control cells, suggesting that ryanodine receptors could play a role in the potentiation of the mGluR‐mediated activation of PLC by Ca2+ release in predepolarized cells.

Nurr1 is required for NMDA receptor-mediated neuronal survival

Background: The mechanism involved in activity-dependent survival of neurons in the central nervous system is not fully understood. Results: Nurr1 is involved in excitatory transmission-dependent survival of glutamatergic neurons by acting downstream CREB and upstream of BDNF. Conclusion: Nurr1 activation mediates activity-dependent survival of glutamatergic neurons. Significance: A novel function of Nurr1 in activity-dependent survival of glutamatergic neurons is reported. NMDA receptor (NMDAR) stimulation promotes neuronal survival during brain development. Cerebellar granule cells (CGCs) need NMDAR stimulation to survive and develop. These neurons differentiate and mature during its migration from the external granular layer to the internal granular layer, and lack of excitatory inputs triggers their apoptotic death. It is possible to mimic this process in vitro by culturing CGCs in low KCl concentrations (5 mm) in the presence or absence of NMDA. Using this experimental approach, we have obtained whole genome expression profiles after 3 and 8 h of NMDA addition to identify genes involved in NMDA-mediated survival of CGCs. One of the identified genes was Nurr1, a member of the orphan nuclear receptor subfamily Nr4a. Our results report a direct regulation of Nurr1 by CREB after NMDAR stimulation. ChIP assay confirmed CREB binding to Nurr1 promoter, whereas CREB shRNA blocked NMDA-mediated increase in Nurr1 expression. Moreover, we show that Nurr1 is important for NMDAR survival effect. We show that Nurr1 binds to Bdnf promoter IV and that silencing Nurr1 by shRNA leads to a decrease in brain-derived neurotrophic factor (BDNF) protein levels and a reduction of NMDA neuroprotective effect. Also, we report that Nurr1 and BDNF show a similar expression pattern during postnatal cerebellar development. Thus, we conclude that Nurr1 is a downstream target of CREB and that it is responsible for the NMDA-mediated increase in BDNF, which is necessary for the NMDA-mediated prosurvival effect on neurons.

Involvement of ETA and ETB receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

This study was performed to characterize the receptor subtypes involved in the endothelin stimulation of phospholipase D (PLD) in rat cortical astrocytes in primary culture. PLD activity was determined by measuring the formation of [32P]phosphatidylbutanol in [32P]orthophosphate prelabelled cells stimulated in the presence of 25 mM butanol. The agonists endothelin‐1 (ET‐1), endothelin‐3 (ET‐3), sarafotoxin 6c (S6c) and IRL 1620 elicited PLD activation in a concentration‐dependent manner. The potencies of ET‐1, ET‐3 and S6c were similar. The maximal effects evoked by the ETB‐preferring agonists, ET‐3, S6c and IRL 1620, were significantly lower than the maximal response to the non‐selective agonist ET‐1. The response to 1 nM ET‐1 was inhibited by increasing concentrations of the ETA receptor antagonist BQ‐123 in a biphasic manner. A high potency component of the inhibition curve (24.2±3.5% of the ET‐1 response) was defined at low (up to 1 μM) concentrations of BQ‐123, yielding an estimated Ki value for BQ‐123 of 21.3±2.5 nM. In addition, the presence of 1 μM BQ‐123 significantly reduced the maximal response to ET‐1 but did not change the pD2 value. Increasing concentrations of the ETB selective antagonist BQ‐788 inhibited the S6c response with a Ki of 17.8±0.8 nM. BQ‐788 also inhibited the effect of ET‐1, although, in this case, two components were defined, accounting for approximately 50% of the response, and showing Ki values of 20.9±5.1 nM and 439±110 nM, respectively. The ET‐1 concentration‐response curve was shifted to the right by 1 μM BQ‐788, also revealing two components. Only one of them, corresponding to 69.8±4.4% of the response, was sensitive to BQ‐788 which showed a Ki value of 28.8±8.9 nM. Rapid desensitization was achieved by preincubation with ET‐1 or S6c. In cells pretreated with S6c neither ET‐3 nor S6c activated PLD, but ET‐1 still induced approximately 40% of the response shown by non‐desensitised cells. This remaining response was insensitive to BQ‐788, but fully inhibited by BQ‐123. In conclusion, endothelins activate PLD in rat cortical astrocytes acting through both ETA and ETB receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ETA and ETB receptors to the ET‐1 response was found to be approximately 20% and 80%, respectively, when ETB receptors were not blocked, and 30–50% and 50–70%, respectively, when ETB receptors were inhibited or desensitized. These results may be relevant to the study of a possible role of PLD in the proliferative effects shown by endothelins on cultured and reactive astrocytes.

Characterization of the metabotropic glutamate receptors mediating phospholipase C activation and calcium release in cerebellar granule cells: Calcium-dependence of the phospholipase C response

In this study we have determined the metabotropic glutamate receptors (mGluRs) involved in the glutamate activation of phospholipase C (PLC) and Ca2+ mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca2+ modulation of the PLC response. Both PLC activation and Ca2+ signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1α and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca2+ for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re‐exposure of cells to Ca2+‐containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca2+ concentration. On the other hand, loading of the intracellular Ca2+ stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca2+ in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca2+ during stimulation and the chelation of cytosolic Ca2+ with BAPTA/AM inhibited both glutamate‐elicited Ca2+ response and PLC activation. Overall, these results indicate that the mGluR‐mediated activation of PLC depends on the presence of extracellular Ca2+ and can be modulated by moderate changes of cytosolic Ca2+. Furthermore, ryanodine reduced PLC stimulation by glutamate in predepolarized cells but not in control cells, suggesting that ryanodine receptors could play a role in the potentiation of the mGluR‐mediated activation of PLC by Ca2+ release in predepolarized cells.

Intracellular Ca2+ stores regulate muscarinic receptor stimulation of phospholipase C in cerebellar granule cells.

Abstract: Muscarinic receptor activation of phosphoinositide phospholipase C (PLC) has been examined in rat cerebellar granule cells under conditions that modify intracellular Ca2+ stores. Exposure of cells to medium devoid of Ca2+ for various times reduced carbachol stimulation of PLC with a substantial loss (88%) seen at 30 min. A progressive recovery of responses was observed following the reexposure of cells to Ca2+‐containing medium (1.3 mM). However, these changes did not appear to result exclusively from changes in the cytosolic Ca2+ concentration ([Ca2+]i), which decreased to a lower steady level (∼25 nM decrease in 1‐3 min after extracellular omission) and rapidly returned (within 1 min) to control values when extracellular Ca2+ was restored. Only after loading of the intracellular Ca2+ stores through a transient 1‐min depolarization of cerebellar granule cells with 40 mM KCl, followed by washing in nondepolarizing buffer, was carbachol able to mobilize intracellular Ca2+. However, the same treatment resulted in an 80% enhancement of carbachol activation of PLC. In other experiments, partial depletion of the Ca2+ stores by pretreatment of cells with thapsigargin and caffeine resulted in an inhibition (18 and 52%, respectively) of the PLC response. Furthermore, chelation of cytosolic Ca2+ with BAPTA/AM did not influence muscarinic activation of PLC in either the control or predepolarized cells. These conditions, however, inhibited both the increase in [Ca2+]i and the PLC activation elicited by 40 mM KCl and abolished carbachol‐induced intracellular Ca2+ release in predepolarized cells. Overall, these results suggest that muscarinic receptor activation of PLC in cerebellar granule cells can be modulated by changes in the loading state of the Ca2+ stores.

Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices

Noradrenaline‐ and ionomycin‐stimulated as well as basal phospholipase D activity from rat hippocampus produced, in the presence of ethanol, two different classes of [32P]phosphatidylethanol (designated I and II), which were separated by thin layer chromatography. Endogenous labeling experiments using 3H‐fatty acids showed that two different classes of phosphatidylcholine, separated by two‐dimensional TLC, one enriched with high incorporation of [3H]arachidonic acid (B) and the other with [3H]myristic acid (A), were the most likely sources for the two classes of phosphatidylethanol. Experiments where individual 32P‐phospholipids extracted from [32P]Pi‐labeled hippocampal slices were incubated with cabbage phospholipase D, in the presence of ethanol, showed that each class of [32P]phosphatidylcholine, i.e. A and B, produced a different band of [32P]phosphatidylethanol, with the same mobility in TLC as phosphatidylethanol II and I, respectively.