FAILURE OF PROLYL-PEPTIDYL ISOMERASE TO MEDIATE CYCLOSPORINE SUPPRESSION OF INTRACELLULAR ACTIVATION SIGNAL GENERATION

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Abstract
Cyclosporine blocks the generation of a cytoplasmic activation protein, activator of DNA replication (ADR). The recent demonstration that cyclophilin is a CsA-sensitive prolyl-peptidyl-isomerase (PPIase), has prompted speculation that CsA immunosuppression is mediated by PPIase interaction with activation signals like ADR. We report that PPIase converts ADR from an inactive to an active form, but the interaction is resistant to CsA. ADR is a sensitive marker of CsA immunosuppression. ADR, extracted from the cytoplasm of PBLs stimulated with PHA, is not detectable in the cytoplasm of resting cells. ADR is quantitated by measuring uptake of 3H/thymidine triphosphate (3H/TPP) into isolated nuclei as a measure of DNA synthesis. The CsA-induced reduction of ADR content mirrored CsA-induced proliferative inhibition in intact cells. CsA concentrations of 1.5, 3, or 4.5 mM reduced T cell proliferation by 26%, 47%, and 58%, and ADR content by 32%, 45%, and 53%, respectively. The ability of PPIase to catalyze the transition of ADR between active and inactive forms was determined by measuring changes in DNA synthesis when 1 microgram/ml PPIase was added to (1) isolated nuclei, (2) nuclei plus ADR, and (3) nuclei plus the cytoplasmic fraction from resting cells. DNA synthesis in isolated nuclei (899 +/- 45 cpm) was unchanged by PPIase (1009 +/- 221 cpm). Addition of PPIase to ADR from activated cells marginally reduced ADR's capacity to trigger 3HTTP incorporation into isolated nuclei (ADR, 4113 +/- 106 cpm; PPIase-treated ADR, 3198 +/- 453 cpm), showing that PPIase cannot reduce ADR activity. However, treatment of resting cell cytoplasm with PPIase increased ADR activity 5-fold (899 +/- 46 vs. 5035 +/- 75 cpm). Addition of 1.5 or 3 mM CsA to resting cytoplasm, primed with PPIase (5035 +/- 75 cpm), resulted in 3HTPP incorporation of 6575 +/- 152 and 5076 +/- 168 cpm, respectively. Thus, PPIase activation of ADR is CsA-resistant. Furthermore, PPIase could not reverse CsA-induced inhibition of ADR. CsA (3 mM) treatment of PHA-stimulated cells rendered proliferation by 30% and ADR by 35%. ADR isolated from cells treated with CsA (9110 +/- 750 cpm) was not increased by treatment with PPIase (9185 +/- 449 cpm). These findings suggest that PPIase converts ADR from an inactive to an active form. However, the mechanism of CsA inhibition of ADR is neither mediated nor overridden by PPIase.

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