Extracellular ubiquitin inhibits beta-AR-stimulated apoptosis in cardiac myocytes: role of GSK-3beta and mitochondrial pathways.
OBJECTIVE
Beta-adrenergic receptor (beta-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVMs) via the activation of glycogen synthase kinase-3beta (GSK-3beta) and mitochondrial pathways. However, beta-AR stimulation induces apoptosis only in a fraction ( approximately 15-20%) of ARVMs. We hypothesized that ARVMs may secrete/release a survival factor(s) which protects 80-85% of cells from apoptosis.


RESULTS
Using two-dimensional gel electrophoresis followed by MALDI TOF and MS/MS, we identified ubiquitin (Ub) in the conditioned media of ARVMs treated with beta-AR agonist (isoproterenol). Western blot analysis confirmed increased Ub levels in the conditioned media 3 and 6 h after beta-AR stimulation. Inhibition of beta1-AR and beta2-AR subtypes inhibited beta-AR-stimulated increases in extracellular levels of Ub, whereas activation of adenylyl cyclase using forskolin mimicked the effects of beta-AR stimulation. Incubation of cells with exogenous biotinylated Ub followed by western blot analysis of the cell lysates showed uptake of extracellular Ub into cells, which was found to be higher after beta-AR stimulation (1.9 +/- 0.4-fold; P < 0.05 vs. control, n = 6). Pre-treatment with Ub inhibited beta-AR-stimulated increases in apoptosis. Inhibition of phosphoinositide 3-kinase using wortmannin and LY-294002 prevented anti-apoptotic effects of extracellular Ub. Ub pre-treatment inhibited beta-AR-stimulated activation of GSK-3beta and c-Jun N-terminal kinase (JNK) and increases in the levels of cytosolic cytochrome c. The use of methylated Ub suggested that the anti-apoptotic effects of extracellular Ub are mediated via monoubiquitination.


CONCLUSIONS
beta-AR stimulation increases levels of Ub in the conditioned media. Extracellular Ub plays a protective role in beta-AR-stimulated apoptosis, possibly via the inactivation of GSK-3beta/JNK and mitochondrial pathways.
Singh
Mahipal
M
Department of Physiology, James H Quillen College of Medicine, James H Quillen Veterans Affairs Medical Center, East Tennessee State University, PO Box 70576, Johnson City, TN 37614, USA.
Roginskaya
Marina
M
Dalal
Suman
S
Menon
Bindu
B
Kaverina
Ekaterina
E
Boluyt
Marvin O
MO
Singh
Krishna
K
D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000235:genetics; D007545:Isoproterenol / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology
D000318:Adrenergic beta-Agonists; D017077:Culture Media, Conditioned; D011943:Receptors, Adrenergic, beta; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D045304:Cytochromes c; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D048031:JNK Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3; D000262:Adenylyl Cyclases; D007545:Isoproterenol