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First published online January 24, 2007
doi: 10.1242/10.1242/jcs.03361

Journal of Cell Science 120, 492-501 (2007)
Published by The Company of Biologists 2007
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Nitric oxide promotes endothelial cell survival signaling through S-nitrosylation and activation of dynamin-2

Ningling Kang-Decker1,*, Sheng Cao1,*,{ddagger}, Suvro Chatterjee1, Janet Yao1, Laurence J. Egan1, David Semela1, Debabrata Mukhopadhyay2 and Vijay Shah1,{ddagger}

1 GI Research Unit, Department of Physiology and Tumor Biology Program, Mayo Clinic, Rochester, MN 55903, USA
2 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55903, USA


Figure 1
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  		Fig. 1. Inhibition of dynamin GTPase abrogates EC survival signals. (A) BAEC transfected with AdGFP or AdK44A were incubated with vehicle or TNF-{alpha}. Cells were collected 24 hours later and prepared for western blot analysis of cleaved caspase-3. Cells transfected with AdK44A showed enhanced TNF-{alpha}-induced BAEC apoptosis as demonstrated by a significant increase in the amount of cleaved caspase-3 from cell lysates. Blots were reprobed with V5 antibody or β-actin for verification of the expression of V5-K44A fusion protein and for protein loading control, respectively (n=3 separate experiments). (B) EC were transfected with dynamin-2 siRNA or scrambled siRNA and cells were harvested for western blot analysis after 48-72 hours or viewed by microscopy for cell morphology. (Top) Prominent morphologic changes and cell death (arrowheads) were detected in EC transfected with dynamin-2 siRNA. (Bottom) EC transfected with dynamin-2 siRNA showed increased levels of cleaved caspase-3 (n=4 separate cell preparations).

 

Figure 2
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  		Fig. 2. Endogenous and exogenous NO promotes survival signals that counterbalance TNF-{alpha}-induced EC apoptosis. (A) BAEC transfected with scrambled siRNA or eNOS-siRNA were incubated with TNF-{alpha} and/or VEGF for 24 hours. Cells were harvested for western blot using antibodies for cleaved caspase-3. VEGF protected cells from TNF-{alpha}-induced apoptosis as shown by the reduced level of cleaved caspase-3 from cell lysates. However, VEGF protection was completely abrogated in cells transfected with eNOS-siRNA. The blot was reprobed with β-actin for a control of protein loading, and for eNOS to confirm siRNA knockdown. (B) BAEC were treated with SNP or TNF-{alpha} or both and collected for protein extraction and western blot. TNF-{alpha} treatment of cells resulted in increased levels of activated caspase-3. Levels of activated caspase-3 were diminished in cells treated with SNP and TNF-{alpha} when compared with cells treated with TNF-{alpha} alone. A representative panel of western blots of triplicate experiments is shown in the top panel, with densitometric analysis of pooled experiments shown in the bottom panel.

 

Figure 3
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  		Fig. 3. NO survival signals are independent of the cGMP-PKG pathway and require dynamin. (A) BAEC transfected with AdGFP or AdK44A were incubated with SNP or TNF-{alpha} or both. Cells were collected 24 hours later and prepared for western blot analysis of cleaved caspase-3. Cells transfected with AdK44A showed enhanced TNF-{alpha}-induced BAEC apoptosis as demonstrated by a significant increase in the amount of cleaved caspase-3 from cell lysates. In the presence of K44A overexpression, SNP-dependent protection from TNF-{alpha}-induced apoptosis was attenuated [representative blot (left panel) and densitometric analysis (right panel); n=5]. Blots were reprobed with anti-V5 antibody or anti β-actin antibody for verification of the expression of V5-K44A fusion protein and for protein loading control, respectively. (B) Tube formation in response to VEGF was assessed by the tube formation assay on Matrigel in EC transfected with AdGFP or AdK44A. Twenty-four hours after transduction, 30,000 cells were seeded in Matrigel-coated wells and the length of endothelial tubes after 12 hours was measured. In GFP-transfected EC, VEGF significantly promoted tube formation (*P<0.05 versus respective basal medium). By contrast, K44A-transfected BAEC failed to form tubes (n=3). Representative phase-contrast images of experimental conditions are shown in the upper panels and compiled data are shown in the graph (lower panel). (C) Left, BAEC were treated with TNF-{alpha} and co-incubated with SNP, 8-Br-cGMP or vehicle for 24 hours. Apoptosis was assessed by Hoechst 33342 stain, and TNF-induced apoptosis was assigned a relative value of 100%. SNP reduced TNF-{alpha}-induced apoptosis whereas 8-Br-cGMP did not (*P<0.05; TNF-{alpha}+SNP versus TNF-{alpha} alone; n=3). Right, BAEC were transfected with adenoviruses encoding GFP (AdGFP) or flag-tagged wild-type PKG (AdPKG), and treated with TNF-{alpha} alone or in combination with 8-Br-cGMP. Cell lysates were prepared 24 hours later for western blot analysis. Neither overexpression of PKG nor combination of PKG with the agonist 8-Br-cGMP protected cells from TNF-{alpha}-induced apoptosis. PKG expression was verified by the presence of Flag epitope.

 

Figure 4
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  		Fig. 4. NO donors increase endocytosis by augmenting dynamin GTPase activity. (A,B) BAEC were preincubated with vehicle or the NO donor GSNO for 20 minutes and then further incubated with fluorescent ligands [fluorescent 488 conjugated transferrin (Tfn) or BODIPY C5-lactosylceramide (LacCer)] for 10 minutes and analyzed by laser scanning cytometry and confocal fluorescence microscopy. Tfn uptake was increased in cells incubated with GSNO as compared with vehicle. Upper panels show representative cell captured by confocal microscopy and lower panels show quantitation by laser scanning cytometry (control R1=6%, GSNO R1=15%). LacCer uptake was also increased in cells incubated with GSNO (control R1=13%, GSNO R1=55%). (C) BAEC were transfected with wild-type dynamin virus or AdK44A prior to GSNO, and Tfn uptake was assayed. AdK44A transduction of EC reduced Tfn uptake and blocked NO-induced increase of Tfn uptake (left: control R1=27%; middle: K44A R1=17%; right: K44A+GSNO R1=18%). (D) Dynamin GTPase activity was measured by thin-layer chromatographic detection of GDP from GTP using recombinant GST-dynamin and a representative experiment is shown. Dynamin incubated with GSNO resulted in an increase in GDP and depletion of GTP, indicating activation of GTPase activity (lane 4). Increased cold GTP competes the conversion from 32P-labeled GTP to GDP (lanes 5-8). Densitometric analysis of the experiment is shown in the lower panel. This experiment was repeated three times with similar results.

 

Figure 5
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  		Fig. 5. NO nitrosylates dynamin. (A) Purified recombinant GST-dynamin, specific subdomains or immunoprecipitated dynamin from cells were incubated with GSNO or vehicle. Alternatively, dynamin was immunoprecipitated from aortic lysates of caveolin wild-type or caveolin-null mice. Nitrosylation of dynamin-2 was assessed by biotin-switch method. S-nitrosylation of immunoprecipitated dynamin from cells is shown (left) [anti-biotin (top panel) and anti-dyn2 (bottom panel)]. Mock represents immunoprecipitation with non-immune serum. Full-length GST dynamin, as well as the GTPase subdomain, showed nitrosylation by DEA-NO. Nitrosylation signal was blocked by preincubation of cells with the reducing agent, ascorbic acid. GST-PRD-domain nitrosylation was not detected. (B) Dynamin nitrosylation was also increased in dynamin-2 immunoprecipitates prepared from aortic lysates of mice genetically deficient in caveolin as compared with wild-type samples (left panel shows a representative autoradiograph and the right panel shows the quantitative change from three independent pooled experiments; *P<0.05). (C) Purified GST-dynamin or the GTPase subdomain were incubated with the NO donor, DEA-NO, or vehicle and then prepared for spectrophotometric analysis. GST-dynamin and the GST-GTPase domain alone incubated with vehicle show a flat curve throughout 200 nm to 450 nm; however, after incubation with GSNO, a characteristic S-nitrosylation peak was detected at ~320 nm.

 

Figure 6
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  		Fig. 6. Nitrosylation of cysteine 86 and cysteine 607 of dynamin-2 regulates NO activation of dynamin and survival signals. BAEC were transfected with retroviral vectors encoding LacZ, wild-type dynamin-2 or mutant dynamin constructs, C24A, C86A or C607A. (A) Cells were preincubated with vehicle or the NO donor (GSNO, 20 minutes) and then LacCer uptake was measured using laser scanning cytometry and confocal fluorescence microscopy. LacCer uptake was increased in cells transfected with LacZ that were incubated with GSNO as compared with vehicle (control R1=7%, GSNO R1=18%). The second panel shows cells transfected with C24A, which also shows increased uptake of LacCer in response to GSNO (control R1=12%, GSNO R1=65%). The remaining two panels show cells transfected with C86A or C607A, which show no significant increase of endocytosis in response to GSNO (control R1=10%, GSNO R1=12% and control R1=11%, GSNO R1=12%, respectively). Upper panels show representative cells captured by confocal microscopy and lower panels show quantitation of histograms by laser scanning cytometry (n=3 independent experimental preparations). (B) Transfected cells were incubated with SNP or TNF-{alpha} or both and collected 24 hours later and prepared for western blot analysis of cleaved caspase-3. Although SNP conferred protection against TNF-{alpha}-induced apoptosis in BAEC that express C24A mutant, overexpression of either C86A or C607A abolished SNP-induced protection from TNF-{alpha}-induced apoptosis. Apoptosis was assessed by measuring levels of cleaved caspase-3 from cell lysates. The blots were probed with anti-β-actin antibody to confirm equal protein loading. The far-right panel western blot shows similar levels of overexpressed dynamin lysates from each of the experimental groups and the increase in dynamin protein levels compared with non-transfected control cell lysates. (C) Purified recombinant GST-dynamin-2 or single-/double-point mutant proteins were incubated with GSNO or vehicle and nitrosylation was assessed by biotin-switch method. Nitrosylation in response to GSNO was detected with the wild-type dynamin protein. An attenuated nitrosylation signal was detected with the C86A and C607A single-point mutant proteins, whereas GSNO-induced nitrosylation was almost undetectable in the C86/C607A double-mutant protein. Quantitative analysis of the level of nitrosylation of the mutant constructs in response to GSNO is shown in the graph to the right.

 

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© The Company of Biologists Ltd 2007