Phosphatidylinositol 3-Akt-kinase-dependent phosphorylation of p21(Waf1/Cip1) as a novel mechanism of neuroprotection by glucocorticoids

The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstrate that corticosterone protects neurons from apoptosis by a mechanism involving the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). In primary cortical neurons, corticosterone leads to a dose- and Akt-kinase-dependent upregulation with enhanced phosphorylation and cytoplasmic appearance of p21(Waf1/Cip1) at Thr 145. Exposure of neurons to the neurotoxin ethylcholine aziridinium (AF64A) results in activation of caspase-3 and a dramatic loss of p21(Waf1/Cip1) preceding apoptosis in neurons. These effects of AF64A are reversed by pretreatment with corticosterone. Corticosterone-mediated upregulation of p21(Waf1/Cip1) and neuroprotection are completely abolished by glucocorticoid and mineralocorticoid receptor antagonists as well as inhibitors of PI3- and Akt-kinase. Both germline and somatically induced p21(Waf1/Cip1) deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21(Waf1/Cip1) suffices to protect neurons from apoptosis. We identify p21(Waf1/Cip1) as a novel antiapoptotic factor for postmitotic neurons and implicate p21(Waf1/Cip1) as the molecular target of neuroprotection by high-dose glucocorticoids

Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

The activation of nuclear factor of activated T cells 5(NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.open3

Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma

Background: Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. Methodology/Principal Findings: We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are.98 % pure, and at least 85 % of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. Conclusions/Significance: We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint an

E2F transcription factors and pRb pocket proteins in cell cycle progression

The E2F-family of transcripion factors exerts fascinating and contrasting functions in transcriptional repression and activation of genes regulating proliferation, apoptosis, and differentiation. E2F is principally regulated by its temporal association with retinoblastoma pocket protein (pRb) family members. In turn, pRb is regulated through phosphorylation by cyclin-dependent kinase (cdk). The activity of cdk is negatively regulated by cdk-inhibitors, exemplified by p16INK4a, p21CIP1, and p27KIP1. Therefore, positive and negative signaling events converge on E2F activity resulting in distinct growth-controling and apoptotic activities. Here we describe the immunocytochemical detection of E2F, genomic DNA, BrdU-incorporation, and mitosis in cardiomyoctes. A detailed protocol is given to illustrate this technique in primary heart muscle cell

Reactive oxygen species induce apoptosis of vascular smooth muscle cell

Apoptosis of vascular smooth muscle cell (VSMC) plays an important role in the genesis of atherosclerosis and restenosis. In order to investigate the role of reactive oxygen species in the induction of VSMC apoptosis, rat VSMCs were treated with glucose oxidase/glucose (GO/G) or diethylmaleate (DEM). The results showed that GO/G and DEM led to VSMC death. Administration of catalase, superoxide dismutase and deferoxamine revealed that H2O2 was the major reactive oxygen species causing cell death, and H2O2O exerted its effect by formation of hydroxyl radical (.OH). GO/G- and DEM-induced VSMC death occurred by apoptosis characterized by "DNA ladders", condensation of nuclei, positive to in situ nick-end labeling and increases in histone-associated DNA fragmentation. This study suggests that H2O2 and its derived form .OH might be related to apoptosis of VSMC in atherosclerosis and restenosis

Differential effect of hydrogen peroxide and superoxide anion on apoptosis and proliferation of vascular smooth muscle cells

Background Proliferation and apoptosis of vascular smooth muscle cells (VSMCs) are two important components of atherosclerosis, restenosis, and hypertension. Although reactive oxygen species have been demonstrated to participate in the pathogenesis of these diseases, their precise involvement has not been fully understood. We hypothesized that different reactive oxygen species exert distinct effects on proliferation and apoptosis of VSMCs. Methods and Results Cultured rat VSMCs were exposed to xanthine oxidase/xanthine (XO/X) or H 2 O 2 -Fe(II). A single exposure to XO/X predominantly resulted in cell proliferation, whereas frequent exposures to high levels of XO/X predominantly resulted in cell death. Administration of superoxide dismutase and catalase revealed that O 2 − but not H 2 O 2 was mitogenic to VSMCs, whereas H 2 O 2 was responsible for VSMC death. Treatment with H 2 O 2 -Fe(II) alone or in the presence of different hydroxyl radical scavengers showed that VSMC death occurred in a dose-dependent manner and was mediated by the formation of hydroxyl radicals. Cell death caused by XO/X or H 2 O 2 -Fe(II) occurred by apoptosis as revealed by condensation of nuclei, appearance of a “DNA ladder,” increases in DNA fragmentation, and positive in situ nick-end labeling. Northern blot analysis indicated that bcl-2 and c- fos but not p53 and c- myc may participate in mediating H 2 O 2 -Fe(II)–induced VSMC apoptosis. Conclusions Different reactive oxygen species exert distinct effects on VSMCs, with O 2 − inducing proliferation and H 2 O 2 causing apoptosis. Thus, reactive oxygen species might participate in atherosclerosis, restenosis, and hypertension in a dual manner by stimulating proliferation and triggering apoptosis of VSMCs. </jats:p