In situ detection of cyclosporin A: evidence for nuclear localization of cyclosporine and cyclophilins.

BACKGROUND

The immunosuppressant cyclosporin A (CsA) forms a trimolecular complex with cyclophilin (CPH) and calcineurins (CN) and inhibits CN phosphatase activity. Inhibition of CN phosphatase by CsA prevents the dephosphorylation of a nuclear factor in the cytosol and its nuclear translocation to the nucleus.

EXPERIMENTAL DESIGN

The intracellular distribution of CPH and CN was investigated in permeabilized Jurkat T lymphocytes and MRC fibroblasts using biochemical techniques and confocal microscopy. The site of CsA binding was identified in situ using a photoaffinity label derivative of CsA followed by immunodetection.

RESULTS

Cyclophilin A (CPH-A) and CN display essentially a cytosolic localization by immunofluorescence, and additional nuclear CPH-A and CN are evidenced by Western blot analysis of purified nuclei and immunofluorescence. By contrast, cyclophilin B (CPH-B) has a punctuate and reticular distribution pattern in cytoplasm, indicating an association with the endoplasmatic reticulum, but its main location is in the nuclear matrix, sparing the nucleolar region. For the in situ detection of CsA binding sites, a photolabile cyclosporine derivative (PL-CS) was used that allowed the detection of covalently bound CsA by Ab. Using the biologically active PL-CS, a punctate cytoplasmatic and nuclear immunoreactivity was obtained, which was specific and competed only with active cyclosporine derivatives. Nuclear CPH-A and -B were labeled by PL-CS, and trimolecular complexes of labeled CPH and CN were obtained by chemically cross-linking nuclear extracts.

CONCLUSIONS

We describe herein the accessibility of CsA to the nucleus, the presence and labeling in situ of nuclear CPH and CN. The current models of CsA action predict that CsA-CPH complexes inhibit CN activity in the cytosol. However, our present findings invite the hypothesis that CPH may capture the drug into the nucleus and target regulatory proteins or transcriptional control elements.