I

Cell Treatment

Fig. 4. Effect of H2O2 on calcineurin-dependent NF-AT activation of luciferase. pNF-Aiy transfected Jurkat cells were either stimulated with ionomycin (2 /xM) and TPA (2 ng/ml) (Stim), left unstimulated (Unstim), stimulated in the presence of 200 ¡iM H202 (Stim + H202), or left unstimulated and treated with 200 fiM H2O2 (Unstim + H202) for 6 hr. Cells were harvested for luciferase assays as described. NF-AT activity is reported as the percentage of relative light units produced by pNF-Aiytransfected Jurkat cells stimulated with ionomycin and TPA alone. Data represent the means of two experiments. [Adapted with permission from T. A. Reiter, R. T. Abraham, M. Choi, and F. Rus-nak, J. Biol. Inorg. Chem. 4,632 (1999), copyright © 1999, Society ofBiological Inorganic Chemistry.]

with ionomycin and TPA (Fig. 4). RLU readings for the positive control vary with each plasmid preparation.

Using this assay, H2O2 was tested for its effect on calcineurin activity in vivo. Transfected cells were treated with ionomycin and TPA and varying concentrations of H2O2 for 6 hr and then harvested for luciferase assays. The activity of calcineurin

Fig. 5. Concentration dependence of H2O2 inhibition of NF-AT activity. Jurkat cells transfected with PNF-AT3 were stimulated with 2 ¡xM ionomycin, TPA (2 ng/ml), and various concentrations of H2O2 for 6 hr. Cells were harvested and the luciferase activity in the lysate was measured. NF-AT activity is shown as the percentage of luciferase activity found in ionomycin/TPA-stimulated, H2O2-untreated cells. Data represent the means of two individual experiments. [Adapted with permission from T. A. Reiter, R. T. Abraham, M. Choi, and F. Rusnak, J. Biol. Inorg. Chem. 4,632 (1999), copyright © 1999, Society of Biological Inorganic Chemistry.]

[H202] fiM

Fig. 5. Concentration dependence of H2O2 inhibition of NF-AT activity. Jurkat cells transfected with PNF-AT3 were stimulated with 2 ¡xM ionomycin, TPA (2 ng/ml), and various concentrations of H2O2 for 6 hr. Cells were harvested and the luciferase activity in the lysate was measured. NF-AT activity is shown as the percentage of luciferase activity found in ionomycin/TPA-stimulated, H2O2-untreated cells. Data represent the means of two individual experiments. [Adapted with permission from T. A. Reiter, R. T. Abraham, M. Choi, and F. Rusnak, J. Biol. Inorg. Chem. 4,632 (1999), copyright © 1999, Society of Biological Inorganic Chemistry.]

can be represented by the luciferase activity in the cell lysate. Using this assay, it can be shown that NF-AT activity is sensitive to exogenous H202 (Fig. 5). The results indicate that H202 inhibits calcineurin-dependent signaling, with a 50% inhibitory concentration (IC50) of ~30-40 ¡iM. Addition of exogenous paraquat, which would generate superoxide as well as H202 via endogenous mechanisms, did not cause a significant decrease in luciferase production.23 We hypothesize that exogenous H202 inactivates calcineurin by altering the redox state of active site iron ion(s).

The pNF-AT3 plasmid is driven by a promoter activated by both NF-AT and AP-1 proteins. Therefore, the inhibitory effect of H202 on pNF-AT3 luciferase production may also be caused by a loss of AP-1 activity in the cell. A second reporter plasmid, pAP-luc, was used to determine whether AP-1 activity was affected

23 T. A. Reiter and F. Rusnak, J. Biol. Inorg. Chem., in press, 2002.

0 0

Post a comment