Extracellular ubiquitin inhibits beta-AR-stimulated apoptosis in cardiac myocytes: role of GSK-3beta and mitochondrial pathways

Abstract

Aims: 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.

Methods and 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.

Conclusion: 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.

Figure 1

Proteome map of conditioned media from CTL and ISO-treated samples. ARVMs were treated with ISO (10 µM) for 3 h. Proteins from the concentrated conditioned media were resolved by 2D gel electrophoresis, stained with SYPRO Ruby fluorescent stain, and photographed. The circled protein was identified as Ub by MALDI TOF and MS/MS. CTL, control; MW, molecular weight ladder (kDa).

Figure 2

β-AR stimulation increases levels of extracellular Ub without affecting membrane integrity. (A) ARVMs were treated with ISO for 3 and 6 h. (B) ARVMs were treated with ISO, or norepinephrine + prazosin (NE/PZ; NE, 10 µM; PZ, 100 nM) or AngII (100 nM) for 3 h. Concentrated conditioned media were subjected to western blot analysis using anti-Ub antibodies. (A) *P < 0.01 vs. CTL; n = 4–7; (B) *P < 0.05 vs. CTL; n = 5. (C) ARVMs were treated with ISO or H₂O₂ for 3 h. The cells were stained with Hoechst 33258 (10 µM) and PI (10 µM) for 10 min, and visualized using fluorescent microscope and photographed. (D) ARVMs were treated with CGP or ICI for 30 min followed by the treatment with ISO for 3 h. To activate adenylyl cyclase, ARVMs were treated with FSK for 3 h. Concentrated conditioned media were subjected to western blot analysis using anti-Ub antibodies. *P < 0.01 vs. CTL; ^#P < 0.05 vs. ISO; n = 6.

Figure 3

β-AR stimulation increases cellular interaction/uptake of Ub. ARVMs were pre-treated with BiotUb (0.5 µg/mL) together with Ub (10 µg/mL) for 30 min before treatment with ISO (3 h). Cell lysates were analysed by western blot using extravidin peroxidase to measure the levels of BiotUb. GAPDH immunostaining indicates protein loading. *P < 0.05 vs. CTL; n = 6.

Figure 4

Extracellular Ub inhibits β-AR-stimulated apoptosis. (A) ARVMs were pre-treated with Ub (10 µg/mL) for 30 min followed by treatment with ISO (Ub + ISO) or pre-treated with ISO for 30 min followed by treatment with Ub (ISO + Ub) for 24 h. The number of apoptotic cells was measured using TUNEL-staining assay. *P < 0.01 vs. CTL; ^#P < 0.01 vs. ISO; n = 3–6. (B) ARVMs were pre-treated with WORT (0.5 µM) for 30 min followed by treatment with Ub (10 µg/mL; 30 min) and ISO (10 µM) for 24 h. The number of apoptotic cells was measured using TUNEL-staining assay. *P < 0.05 vs. CTL; ^#P < 0.05 vs. ISO; ^&P < 0.05 vs. Ub + ISO; n = 3–5. (C) ARVMs were pre-treated LY for 30 min followed by treatment with Ub (10 µg/mL; 30 min) and ISO (10 µM) for 24 h. The number of apoptotic cells was measured using TUNEL-staining assay. *P < 0.05 vs. CTL; ^#P < 0.05 vs. ISO; ^&P < 0.05 vs. Ub + ISO; n = 4–6.

Figure 5

Extracellular Ub inhibits β-AR-stimulated activation of GSK-3β and JNKs and increases in the levels of cytosolic cytochrome c. (A and B) ARVMs were treated with Ub (10 µg/mL) for 30 min before treatment with ISO (15 min). Cell lysates were analysed by immune-complex kinase assay to measure GSK-3β activity (A) or by western blot using phospho-specific anti-JNK antibodies (B). *P < 0.05 vs. CTL; n = 3; ^#P < 0.05 vs. ISO; n = 3–5. (C) ARVMs were infected with adenoviruses expressing constitutively active GSK-3β (S9A-GSK) for 48 h. Cell lysates were analysed by western blot using phospho-specific anti-JNK antibodies. GAPDH immunostaining indicates protein loading. *P < 0.05 vs. CTL. (D) ARVMs were pre-treated with Ub (10 µg/mL) for 30 min followed by treatment with ISO (10 µM, 6 h). Cytosolic cytochrome c levels were measured using western blot analysis. GAPDH immunostaining indicates protein loading. *P < 0.05 vs. CTL, ^#P < 0.05 vs. ISO; n = 4.

Figure 6

Anti-apoptotic effects of extracellular Ub are mediated via monoubiquitination. ARVMs were pre-treated with MeUb (10 µg/mL) for 30 min followed by treatment with ISO (24 h). The numbers of apoptotic cells were measured using TUNEL-staining assay. *P < 0.05 vs. CTL; ^#P < 0.05 vs. ISO; n = 4.