E FFECTS OF OXIDANTS ON CALCINEURIN - DEPENDENT SIGNALING IN INTACT CELLS

3.5.1. INHIBITION OF CALCINEURIN PHOSPHATASE ACTIVITY AND CALCINEURIN-DEPENDENT

NFAT1 DEPHOSPHORYLATION BY H2O2 IN JURKAT CELLS.

Having investigated the effects of oxidants on the activity of isolated calcineurin, we went on to test whether oxidative stress affects calcineurin in intact cells. Human Jurkat T-lymphoma cells provide a well-established model for calcineurin-dependent signaling in lymphocytes. In T-cells, activation of the T-cell receptor followed by elevation of intracellular calcium levels causes increase of calcineurin activity. Active calcineurin dephosphorylates proteins of the NFAT transcription factor family, which in their dephosphorylated state translocate into the nucleus and activate transcription of a number of genes involved in lymphocyte activation, including cytokines IL-2 or IL-4. We could follow the functional status of different steps of the calcineurin-NFAT cascade by monitoring calcineurin activity in Jurkat cell lysates, phosphorylation status of NFAT1 protein and NFAT transcriptional activity using a β-galactosidase reporter assay (courtesy of Prof. U. Ruegg, University of Lausanne, Switzerland). Several agents were used to model conditions of cellular oxidative stress. H2O2 is a most commonly used oxidant for treatment of cells, and it is considered to reflect the conditions of oxidative stress in general. Thus, we tested the effects of treatment of Jurkat cells with H2O2 on calcineurin activity in cellular lysates. Application of H2O2 dose-dependently inhibited calcineurin activity in Jurkat cells (Fig. 3.5.1).

Figure 3.5.1. H₂O₂ treatment of Jurkat cells blocks calcineurin activity of cellular extracts.

Jurkat cells (10⁶/ml) were treated for 30 min with indicated concentrations of H2O2 or with 1 µM cyclosporin A (CsA). Calcineurin activity of cellular extracts was measured with ³²P-RII peptide.

As a control, treatment of the cells with cyclosporin A (1µM) almost completely blocked calcineurin activity in cellular extracts. In contrast, incubation of Jurkat cells for 30 min with XO/hypoxanthine caused no significant inhibition of calcineurin activity, indicating that superoxide generated extracellularly is unlikely to reach the calcineurin pool within the cell. The data of activity tests were re-confirmed by monitoring the phosphorylation status of calcineurin target protein, NFAT1, on

Western blots. Dephosphorylated NFAT1 has higher mobility on SDS-PAGE and therefore is shifted towards apparently lower molecular weight than the native, heavily phosphorylated form. Fig. 3.5.2. shows the results of such electrophoretic mobility tests.

Figure 3.5.2. H₂O₂ inhibits calcineurin-mediated NFAT1 in ionomycin-stimulated Jurkat cells. Jurkat cells (10⁶/ml) were treated for 30 min with indicated concentrations of H2O2 or with 1 µM cyclosporin A (CsA) and stimulated for 5 min with 2 µM ionomycin. Cell extracts were subjected to Western blot with anti-NFAT1 antibody. Lanes: 1, control; 2, ionomycin; 3, 1 µM CsA; 4, 0.2 mM H2O2; 5, 0.5 mM H2O2; 6, 1 mM H2O2.

Elevation of intracellular calcium levels in Jurkat cells induced by ionomycin caused calcineurin activation and NFAT1 dephosphorylation, which was blocked by cyclosporin A.

This activation-induced mobility shift was also dose-dependently inhibited by H2O2, with 1 mM completely blocking NFAT1 dephosphorylation. Thus, inhibition of calcineurin activity by oxidants results in consequent increase of phosphorylation of calcineurin intracellular targets.

3.5.2. CALCINEURIN-DEPENDENT NFAT TRANSCRIPTIONAL ACTIVITY IN JURKAT CELLS IS INHIBITED BY OXIDANTS.

NFAT transcriptional activity can be monitored by using genetically engineered reporter plasmids, where expression levels of a specific reporter protein are under transcriptional control of a gene promoter element targeted by the transcription factor, which activity is to be investigated. We obtained the possibility to use a Jurkat line stably transfected with a β-galactosidase reporter plasmid under control of a promoter from IL-2 distal site, which has three NFAT-binding sites. By measuring β-galactosidase activity after cell stimulation with a mix of phorbol ester (PMA) and phytohemagglutinin (PHA) one has a means to monitor NFAT transcriptional activity under different conditions. Several oxidants were tested for their effects on the activity of NFAT-dependent reporter in Jurkat cells. As

expected, H2O2 suppressed reporter activity at concentrations 50 µM and higher (Fig. 3.5.3).

Similarly, 200 µM organic hydroperoxide tert-butylhydroperoxide (t-BuOOH) inhibited NFAT-dependent transcription.

Figure 3.5.3. Effect of peroxides on NFAT-dependent transcriptional activity in Jurkat cells. Jurkat cells stably transfected with β-galactosidase reporter plasmid under control of IL-2 promoter were stimulated with 20 ng/ml PMA and 1µg/ml PHA for 20 h in the presence of indicated concentrations of H2O2 or 200 µM tert-butylhydroperoxide (t-BuOOH). At the end of incubation β-galactosidase activity of cell lysate was measured.

When applied extracellularly, XO also reduced reporter activity to background levels. This effect was, however, completely H2O2-dependent, since it was blocked by the presence of catalase, but not SOD, in the extracellular medium (Fig. 3.5.4).

Figure 3.5.4. Effect of XO on NFAT-dependent transcriptional activity in Jurkat cells. Jurkat cells stably transfected with β-galactosidase reporter plasmid under control of IL-2 promoter were stimulated with 20 ng/ml PMA and 1µg/ml PHA for 20 h in the presence of indicated concentrations XO (mU/ml) and 100 µM hypoxanthine. When indicated, SOD (200 U/ml) or catalase (Cat, 200 U/ml) were added together with XO. At the end of incubation

β-galactosidase activity of cell lysate was measured.

To induce intracellular superoxide generation the redox-cycling compounds paraquat and DMNQ were used. These compounds undergo cycles of univalent oxidation/reduction by molecular oxygen catalyzed by cellular NADH/NADPH-dependent oxidoreductases, generating significant amounts of superoxide. Incubation of Jurkat cells with 10 µM DMNQ resulted in 80% inhibition of reporter activity, although 5 µM caused only 20% decrease.

Paraquat instead, even in high concentrations (1 mM) inhibited only 30% of the reporter activity (Fig. 3.5.5). Neither catalase, nor SOD could block the effect of 10 µM DMNQ when added extracellularly (data not shown).

Figure 3.5.5. Effect of redox-cycling agents on NFAT-dependent transcriptional activity in Jurkat cells. Jurkat cells stably transfected with β-galactosidase reporter plasmid under control of the IL-2 promoter were stimulated with 20 ng/ml PMA and 1µg/ml PHA for 20 h in the presence of indicated concentrations of paraquat (PQ) or DMNQ. At the end of incubation β-galactosidase activity of cell lysate was measured.

Therefore it is not clear whether the inhibition caused by DMNQ is superoxide-dependent.

Thus, when applied from outside of the cell, hydrogen peroxide seems to be the most effective oxidant for calcineurin in T-lymphocytes.

3.5.3. STIMULATION OF RESPIRATORY BURST IN MACROPHAGES CAUSES INHIBITION OF ENDOGENOUS CALCINEURIN ACTIVITY.

Although extracellular generation of O2- was not able to influence calcineurin activity in Jurkat cells, the high sensitivity of calcineurin to superoxide in vitro indicated that intracellular O2- generation should under some conditions cause calcineurin inhibition. The respiratory burst in activated macrophages could provide such conditions. Upon stimulation, the NADPH oxidase system of macrophages generates high amounts of superoxide, most of which is compartmentalized in the phagocytic vacuole. Part of it, however, could leak into cytosol where calcineurin is located. Since macrophage activation is accompanied by elevation of cytosolic calcium levels, such conditions favor oxidative inactivation of calcineurin. To test the effects of macrophage activation on calcineurin activity, we incubated the mouse macrophage line RAW 264.7 with phorbol myristate acetate (PMA), an optimal treatment for respiratory burst stimulation. As shown on Fig. 3.5.6. 500 nM PMA caused

~50% inhibition of calcineurin activity in cell extracts.

Figure 3.5.6. Respiratory burst inhibits calcineurin activity in RAW 264.7 macrophages. RAW 264.7 cells (10⁶/ml) were incubated for 30 min with 500 nM PMA alone or together with 50 µM diphenyleniodonium (DPI), and calcineurin activity of cellular extracts was measured with ³²P-RII peptide.

This inhibition was prevented by co-treatment of the cells with diphenyleniodonium (DPI), an inhibitor of the respiratory burst oxidase system. These results suggest that intracellular superoxide generation is capable of inhibiting endogenous calcineurin activity.