Dexamethasone and triamcinolone acetonide accumulation in mouse fibroblasts is differently modulated by the immunosuppressants cyclosporin A, FK506, rapamycin and their analogues, as well as by other P-glycoprotein ligands.

摘要

In mouse fibroblasts (LMCAT cells) stably transfected with the reporter gene chloramphenicol acetyl transferase under the control of the mouse mammary tumor virus promoter (MMTV-CAT), cyclosporin A (CsA), FK506, and rapamycin (Rap) at micromolar concentrations potentiate dexamethasone- (Dex) induced CAT gene activity in a dose-dependent way (Renoir J.-M., Mercier-Bodard C., Hoffmann K., Le Bihan S., Ning Y. M., Sanchez E. R., Handschumacher R. E. and Baulieu E. E., Proc. Natl. Acad. Sci. U.S.A., 92, 1995, 4977-4981). In this work, we used LMCAT and 1471.1 cells, another mouse fibroblast cell line stably transfected with the MMTV-CAT construct, and found that exposure to immunosuppressants affected steroid-induced transcription differently. Indeed, all immunosuppressants, including inactive analogues, potentiated not only Dex- but also TA-induced CAT gene expression in LMCAT cells. The extent of this potentiation was 3 times lower for TA than for Dex. These immunosuppressants have no effect in 1471.1 cells. In addition, no difference of glucocorticosteroid affinity for the GR was observed in 1471.1 cells, in contrast to LMCAT cells. In both cell lines, the drugs tested increased [3H] Dex and [3H] TA (although to a lesser extent) accumulation. Since it is known that immunosuppressants can reverse the membrane Phospho-glycoprotein (P-gp) activity responsible for an active efflux of small hydrophobic molecules from numerous cell types, we therefore measured the relative efficiency of other P-gp ligands (including vinca alkaloids and the inactive CsA analogue, PSC833), on [3H] Dex and [3H] TA accumulation. In both cell lines, and depending on the drugs, reversal of Dex export was more pronounced than that of TA export (approximately 11 times in LMCAT and approximately 2 times in 1471.1 cells). However, the antiprogestin/antiglucocorticosteroid RU 38 486 and its 17beta derivatives RU 49 953 which does not bind to GR, both identified as strong reversal molecules of P-gp activity, had respectively, no and a strong inhibiting effect on steroid accumulation in both cell lines. These results suggest that a mechanism resembling but different from P-gp can modulate steroid entry into these mouse fibroblasts. This is confirmed by the failure to demonstrate the presence of P-gp by immunoprecipitation and Western blot experiments in membrane preparations from both cell lines. From these data, we conclude: (i) that the two synthetic GR ligands do not accumulate similarly in mouse fibroblasts, (ii) that RU 49 953 increases steroid efflux, in contrast to other agents known to reverse P-gp activity (iii) that cellular entry and export of Dex and TA can be modulated by membrane efflux mechanism(s), different from P-gp, and (iiii) that immunosuppressant potentiation of Dex- and TA-induced CAT activity involves such a mechanism in LMCAT cells. In 1471.1 cells, the lack of any enhancing effect upon steroid-induced transcription of all the drugs tested, although they all increase steroid accumulation, suggests involvement of immunosuppressant-influenced factor(s) acting downstream from steroid entry, in the hormone receptor-mediated transcription pathway(s).