Calcineurin enhances MEF2 DNA binding activity in calcium-dependent survival of cerebellar granule neurons.

Myocyte enhancer factor 2 (MEF2) has been shown recently to be necessary for mediating activity-dependent neuronal survival. In this study, we show that calcium signals regulate MEF2 activity through a serine/threonine phosphatase calcineurin. In cultured primary cerebellar granule neurons, the electrophoretic mobility of MEF2A protein was sensitive to the level of extracellular potassium chloride (KCl) and depolarizing concentrations of KCl led to hypophosphorylation of the protein. The specific inhibitors of calcineurin cyclosporin A (CsA) and FK506 could overcome KCl-dependent MEF2A hypophosphorylation. The effects of CsA and FK506 were KCl specific as they had little effect on MEF2A phosphorylation when granule neurons were cultured in the presence of full media. Hyperphosphorylation of MEF2A led to the loss of its DNA binding activity as determined by DNA mobility shift assay. Consistent with this, CsA/FK506 also inhibited MEF2-dependent reporter gene expression. These findings demonstrate that regulation of MEF2A by calcium signals requires the action of protein phosphatase calcineurin. By maintaining MEF2A in a hypophosphorylated state, calcineurin enhances the DNA binding activity of MEF2A and therefore maximizes its transactivation capability. The identification of MEF2 as a novel target of calcineurin may provide in part a biochemical explanation for the therapeutic and toxic effects of immunosuppressants CsA and FK506.     

Chloride conductance is required for the protein kinase A and Rac1-dependent phosphorylation of moesin at Thr-558 by KCl in PC12 cells

Moesin is a member of the ERM family, a family of cross-linkers between the plasma membrane and the actin cytoskeleton, which are known to be activated by phosphorylation. Previously, we reported the RhoA and Rho kinase-dependent phosphorylation of moesin at Thr-558 in hippocampal neuronal cells by glutamate. Here we studied the induction of moesin phosphorylation by KCl (60 mm) in PC12 cells. Moesin phosphorylation at Thr-558 was increased after 2 min of KCl treatment, peaked at 5 min, and returned to the basal level by 60 min. KCl also activated Rac1, but not RhoA, in PC12 cells, and KCl-induced moesin phosphorylation was suppressed in dominant negative Rac1 (N17 Rac1)-expressed cells. The inhibition of protein kinase A (PKA), known as an upstream kinase of Rac1, abolished Rac1 activation and moesin phosphorylation by KCl. Interestingly, the phosphorylation of moesin by KCl was independent of KCl-induced membrane depolarization and calcium influx but was dependent on KCl-induced chloride conductance. 60 mm KCl induced chloride conductance in PC12 cells, and pretreatment with Cl- channel blocker abolished Rac1 activation and moesin phosphorylation by KCl. These results suggest that the phosphorylation of moesin at Thr-558 in PC12 cells by KCl treatment is PKA- and Rac1-dependent and that KCl-induced chloride conductance is involved in the activation of this signaling system.     

Endogenous and exogenous fibroblast growth factor 2 support survival of chick retinal neurons by control of neuronal neuronal bcl-x(L) and bcl-2 expression through a fibroblast berowth factor receptor 1- and ERK-dependent pathway

Fibroblast growth factor (FGF) 2 is a survival factor for various cell types, including retinal neurons. However, little is understood about the molecular bases of the neuroprotective role of FGF2 in the retina. In this report, FGF2 survival activity was studied in chick retinal neurons subjected to apoptosis by serum deprivation. Exogenous FGF2 supported neuronal survival after serum deprivation and increased neuronal bcl-x(L) and bcl-2 expression, through binding to its receptor R1 (FGF-R1), and subsequent extracellular signal-regulated kinase (ERK) activation. Endogenous FGF2 was transiently overexpressed after serum deprivation. Its down-regulation by antisense oligonucleotides and blockade of its signaling pathway (binding to FGF-R1, tyrosine phosphorylation, and ERK inhibition) decreased bcl-x(L) and bcl-2 levels and and enhanced apoptosis, suggesting that endogenous FGF2 supported neuronal survival through a pathway similar to that of exogenous FGF2. This pathway may serve to up-regulate, or maintain, bcl-x(L) and bcl-2 levels that normally decrease during the onset of apoptosis. Indeed, long-term ERK activation and high bcl-x(L) levels are necessary for the survival activity of both exogenous and endogenous FGF2. Because FGF2 is upregulated following retinal injury in vivo, we suggest that an injury-stimulated autocrine/paracrine FGF2 loop may serve to maintain high levels of survival proteins, such as Bcl-x(L), through ERK activation in retinal neurons.     

Withdrawal of Survival Factors Results in Activation of the JNK Pathway in Neuronal Cells Leading to Fas Ligand Induction and Cell Death

The JNK pathway modulates AP-1 activity. While in some cells it may have proliferative and protective roles, in neuronal cells it is involved in apoptosis in response to stress or withdrawal of survival signals. To understand how JNK activation leads to apoptosis, we used PC12 cells and primary neuronal cultures. In PC12 cells, deliberate JNK activation is followed by induction of Fas ligand (FasL) expression and apoptosis. JNK activation detected by c-Jun phosphorylation and FasL induction are also observed after removal of either nerve growth factor from differentiated PC12 cells or KCl from primary cerebellar granule neurons (CGCs). Sequestation of FasL by incubation with a Fas-Fc decoy inhibits apoptosis in all three cases. CGCs derived from gld mice (defective in FasL) are less sensitive to apoptosis caused by KCl removal than wild-type neurons. In PC12 cells, protection is also conferred by a c-Jun mutant lacking JNK phosphoacceptor sites and a small molecule inhibitor of p38 mitogen-activated protein kinase and JNK, which inhibits FasL induction. Hence, the JNK-to-c-Jun-to-FasL pathway is an important mediator of stress-induced neuronal apoptosis.     

Phosphorylation of IkappaB-beta is necessary for neuronal survival

Cerebellar granule neurons undergo apoptosis when switched from culture medium containing depolarizing levels of potassium (high potassium or HK) to nondepolarizing medium (low potassium or LK). We showed that in healthy neurons maintained in HK medium, IkappaB-beta is phosphorylated at a novel site, Tyr-161. LK-induced neuronal apoptosis is accompanied by a decrease in the extent of IkappaB-beta phosphorylation at this residue. Tyr-161 shares similarity to the consensus sequence for phosphorylation by the nonreceptor tyrosine kinases Abl and Arg. Arg phosphorylates Tyr-161 differentially in vitro, and LK treatment does cause a down-regulation of Arg activity. Moreover, treatment of neurons with two structurally distinct and highly selective Abl inhibitors, PD173955 and Gleevec, blocks HK-induced phosphorylation of IkappaB-beta at Tyr-161 and induces neuronal apoptosis. Overexpression of wild-type IkappaB-beta blocks LK-induced apoptosis, but this effect is abolished when Arg is pharmacologically inhibited. On the other hand, forced overexpression of IkappaB-beta in which Tyr-161 is mutated inhibits survival in HK demonstrating the importance of this residue to neuronal survival. Phosphorylation of IkappaB-beta enhances its association with p65/RelA causing an increase in NF-kappaB DNA binding activity. Our results identified IkappaB-beta phosphorylation as a key event in neuronal survival and provided a mechanism by which this is mediated.