Cell survival and death play critical roles in tissues composed of
Cell survival and death play critical roles in tissues composed of post-mitotic cells. disruption of Epac exerted a protective effect on neuronal apoptosis -amyloid protein, sialoglycopeptide, low potassium-induced neurotoxicity) (2,C4), although there have been 945595-80-2 supplier several reports that dopamine or prostanoid receptor-mediated cAMP production induces neurotoxicity (5, 6). -Adrenergic receptor signaling, on the other hand, promotes apoptosis in cardiac myocytes, resulting in heart failure (7, 8). Therefore, the model proposing that cAMP signaling plays a protective role in neuronal cells but a deteriorative role in myocardial cells is well accepted. Most studies that have demonstrated the effect of cAMP signaling on apoptosis have focused primarily on protein kinase A (PKA),2 a classic target molecule of cAMP. Recent studies involving cAMP signaling have focused instead on Epac, an exchange protein activated by cAMP that has been identified as a new target of cAMP, independent of PKA (9). Epac has been found to regulate a variety of cellular processes, including cell proliferation, migration, secretion, and differentiation (10). It has been demonstrated that Epac either alone or with PKA plays a protective role in immune cells against apoptosis (11, 12). In post-mitotic cells such as neurons and cardiac myocytes, however, the role of Epac in apoptosis has not been reported. To date two isoforms of Epac have been identified, Epac1 and Epac2 (9); they differ in that Epac2 contains a second binding site for cAMP. It has recently been reported that there is an up-regulation of Epac1 mRNA in Alzheimer disease (13) and an up-regulation of Epac1 protein expression in rats with inflamed neurons (14), implicating that cAMP signaling may not always play a protective role in neurons. The change in the Epac1 expression pattern has also been demonstrated in other cell types (heart, vasculature, kidney, and lung) (15,C18). The stoichiometry of Epac, especially of Epac1, and that of PKA might be changed in several diseases, including neuronal and cardiac disorders; this could lead to the various effects of cAMP signaling on cell death. Through experiments using Epac- or PKA-selective cAMP analogs and overexpression of Epac1 and the PKA catalytic subunit and Epac1-deficient mice, the present study demonstrates that cAMP signaling no longer increases neuronal cell viability when Epac is selectively activated: instead, cAMP signaling induces apoptosis through increasing Bcl-2 interacting member protein (Bim) expression. Our findings also suggest that the selective inhibition of Epac signaling may become a therapeutic strategy in the treatment of neurodegenerative diseases. EXPERIMENTAL PROCEDURES Antibodies and Reagents 8-labeling of fragmented DNA in cultured cortical neurons and cardiac myocytes was performed using the DeadEndTM fluorometric TUNEL system (Promega, Madison, WI) according to the manufacturer’s instructions. Cells were incubated with the presence or absence of pMe-cAMP or Bnz-cAMP for 48 h, fixed with 4% paraformaldehyde for 25 min, and then incubated with 0.2% Triton X-100 for 5 min. The cells were equilibrated with a buffer consisting of 200 mm potassium cacodylate (pH 6.6), 945595-80-2 supplier 25 mm Tris-HCl (pH 8.0), 0.2 mm dithiothreitol, 0.25 mg/ml bovine serum albumin, and 2.5 mm cobalt chloride at room temperature for 10 min followed by 60 min of incubation with a terminal deoxynucleotidyltransferase reaction buffer containing 100 m dATP, 5 m fluorescein-12-dUTP, 10 mm Tris-HCl (pH 7.6), 1 mm EDTA, and 40 m terminal deoxynucleotidyltransferase enzyme at 37 C. DNAs were stained with DAPI (4, 6-diamidino-2 phenylindole). The percentage of the total cells that were TUNEL-positive was determined in a blinded manner. Approximately 2000C3000 cells in 10 945595-80-2 supplier randomly selected fields from each sample were counted. For detection of apoptosis in brain tissues from WT or Epac1 KO mice, deparaffinized tissue sections were treated with 20 g/ml proteinase K and 50 mm EDTA in 100 mm Tris-HCl (pH 8.0). The sections were fixed with 4% paraformaldehyde for 15 min at room temperature and then subjected to the equilibration step in the procedures described above. Analysis of DNA Fragmentation by Enzyme-linked Immunosorbent Assay Histone-associated DNA fragments were quantified using the Cell Death Detection enzyme-linked immunosorbent assay kit (Roche Diagnostics) according to the manufacturer’s instructions. After MAP2K2 cortical neurons and cardiac.