Fig 1.
β-AR stimulation induces post-translational modifications of NF2 in ARVMs.
ARVMs were treated with ISO (10 μM) for 15 mins. Total cell lysates were analyzed by western blot using phospho-specific anti-NF2 antibodies. This antibody recognized ~70 kDa (phosphorylated NF2; A) and ~100 kDa (phosphorylated and sumoylated NF2; B) proteins. The lower panels exhibit the mean data normalized to total NF-2 (T-NF2) or GAPDH, *p<0.05 vs CTL; n = 3–7. (C) To confirm NF2 sumoylation, cell lysates were immunoprecipitated with anti-NF2 antibodies. Immunoprecipitates were analyzed by western blot using anti-SUMO-1 antibodies. Panel C exhibits quantitative increase in NF2 sumoylation in response to ISO. *p<0.05 vs CTL; n = 3.
Fig 2.
Adenoviral mediated expression of WT-NF2 induces post-translational modifications of NF2.
ARVMs were infected with adenoviruses expressing WT-NF2 or GFP for 48 h. Cell lysates were analyzed by western blot using phospho-specific anti-NF2 antibodies. (A) Phosphorylated (~70 kDa) NF2; (B) Phosphorylated and sumoylated (~100 kDa) NF2. The lower panels exhibit the mean data normalized to GAPDH, *p<0.05 vs CTL; n = 6–7.
Fig 3.
β-AR stimulation increases post-translational modifications of NF2 in nuclear and cytosolic fractions.
ARVMs were treated with ISO for 15 mins. The cytosolic (CF) and nuclear fractions (NF) were analyzed by western blot using phospho-specific anti-NF2 antibodies. The lower panels exhibit the mean data normalized to GAPDH (CF) or PARP (NF), *p<0.05 vs CTL; n = 5–7.
Fig 4.
Involvement of β-AR subtypes, adenylyl cyclase and PKA in post-translational modifications of NF2.
(A) ARVMs in CGP+ISO, ICI+ISO and H89+ISO groups were pretreated with CGP, ICI and H89, respectively, for 30 min prior to ISO treatment for 15 min. (B) ARVMs were treated with forskolin (FSK), an adenylyl cyclase activator, for 15 min. Cell lysates were analyzed by western blot using phospho-specific anti-NF2 antibodies. The lower panels exhibit the mean data normalized to GAPDH. *p<0.05 vs. CTL; $p<0.05 vs. ISO; n = 6–9.
Fig 5.
β-AR stimulation and adenoviral-mediated expression of NF2 increase phosphorylation of MST1/2 in ARVMs.
ARVMs were treated with ISO for 15 min (A) or infected with adenoviruses expressing WT-NF2 or GFP for 48 h (B). Total cell lysates were analyzed by western blot using phosho-specific anti-MST1/2 antibodies. The lower panels exhibit the mean data normalized to GAPDH, *p<0.05 vs CTL or GFP; n = 5.
Fig 6.
Stimulation of β-AR and adenoviral-mediated expression of NF2 increase YAP phosphorylation.
ARVMs were treated with ISO for 15 min (A) or infected with adenoviruses expressing WT-NF2 or GFP for 48 h (B). Total cell lysates were analyzed by western blot using phospho-specific anti-YAP antibodies. There was no significant change in total YAP expression following ISO treatment. Therefore, data was normalized to GAPDH. The lower panels exhibit the mean data normalized to GAPDH, *p<0.05 vs CTL or GFP; n = 3–7.
Fig 7.
Knockdown of NF2 using siRNA decreases β-AR-stimulated increase in NF2 and YAP phosphorylation.
H9C2 cells were transfected with NF2 siRNA for 48 h followed by treatment with ISO for 15 min. Cell lysates were analyzed by western blot using anti-NF2 (A), phospho-specific anti-NF2 (B) and phospho-specific anti-YAP (C) antibodies. The lower panels exhibit the mean data normalized to GAPDH, *P<0.05 vs.CTL; $p<0.05 vs. ISO; n = 3.
Fig 8.
Knockdown of NF2 inhibits β-AR-stimulated apoptosis, while adenoviral-mediated expression of WT-NF2 induces apoptosis.
(A) ARVMs were transfected with NF2 siRNA or negative control siRNA (neg siRNA) for 24 h followed by treatment with ISO for 24 h. Apoptosis was measured using TUNEL assay. *p<0.05 vs CTL; $p<0.05 vs ISO or neg siRNA+ISO; n = 3–4. (B) ARVMs were infected with adenoviruses expressing WT-NF2 or GFP for 48 h. Apoptosis was measured using TUNEL assay. *p<0.05 vs GFP; n = 3–4.
Fig 9.
Adenoviral-mediated expression of NF2 activates mitochondrial death pathway in ARVMs.
ARVMs were infected with adenoviruses expressing WT-NF2 or GFP for 48 h. Cell lysates were analyzed by western blots using phospho-specific anti-JNKs (A), anti-Bax (B), and anti-Bcl2 antibodies (data not shown), while cytosolic fractions were analyzed by western blot using anti-cytochrome c (D) antibodies. The lower panels exhibit the mean data normalized to total JNKs (T-JNKs) or GAPDH. Fig 9 (C) represents Bcl2/Bax ratio. *p<0.05 vs GFP; n = 4–6.
Fig 10.
Proposed schematic representation of signaling pathway involved in β-AR-stimulated increase in post-translational modifications of NF2.
β-AR-stimulation using isoproterenol increases phosphorylation and sumoylation of NF2. This pathway may involve β1-AR and cAMP/PKA pathway since β1-AR antagonism and H89 inhibit β-AR-stimulated increase in NF2 phosphorylation and sumoylation. Direct activation of adenylyl cyclase mimics the effects of β-AR-stimulation on NF2 phosphorylation and sumoylation. β-AR-stimulation increases levels of phosphorylated and sumoylated NF2 in the nucleus. β-AR-stimulation increases phosphorylation of MST1/2 and YAP, downstream targets of NF2. Expression of WT-NF2 using adenoviruses stimulates mitochondrial death pathway by activating JNKs, increasing Bax expression and enhancing cytosolic levels of cytochrome c.