DEXAMETHASONE AND TRIAMCINOLONE ACETONIDE ACCUMULATION IN MOUSE FIBROBLASTS IS DIFFERENTLY MODULATED BY THE IMMUNOSUPPRESSANTS CYCLOSPORINE-A, FK506, RAPAMYCIN AND THEIR ANALOGS, AS WELL AS BY OTHER P-GLYCOPROTEIN LIGANDS

In mouse fibroblasts (LMCAT cells) stably transfected with the reporte r gene chloramphenicol acetyl transferase under the control of the mou se mammary tumor virus promoter (MMTV-CAT), cyclosporin A (CsA), FK506 , and rapamycin (Rap) at micromolar concentrations potentiate dexameth asone- (Dex) induced CAT gene activity in a dose-dependent way (Renoir J.-M., Mercier-Bodard C., Hoffmann K., Le Bihan S., Ning Y. M., Sanch ez E. R., Handschumacher R. E. and Baulieu E. E., Proc. Natl. Acad. Sc i. 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 th e MMTV-CAT construct, and found that exposure to immunosuppressants af fected steroid-induced transcription differently. Indeed, all immunosu ppressants, including inactive analogues, potentiated not only Dex- bu t also TA-induced CAT gene expression in LMCAT cells. The extent of th is potentiation was 3 times lower for TA than for Dex. These immunosup pressants have no effect in 1471,1 cells. In addition, no difference o f glucocorticosteroid affinity for the GR was observed in 1471.1 cells , in contrast to LMCAT cells. In both cell lines, the drugs tested inc reased [H-3] Dex and [H-3] TA (although to a lesser extent) accumulati on. Since it is known that immunosuppressants tan 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 vin ca alkaloids and the inactive CsA analogue, PSC833), on [H-3] Dex and [H-3] TA accumulation. In both cell lines, and depending on the drugs, reversal of Dex export was more pronounced than that of TA export (si milar to 11 times in LMCAT and similar to 2 times in 1471.1 cells). Ho wever, the antiprogestin/antiglucocorticosteroid RU 38 486 and its 17 beta derivatives RU 49 953 which does not bind to GR, both identified as strong reversal molecules of P-gp activity, had respectively, no an d a strong inhibiting effect on steroid accumulation in both cell line s. These results suggest that a mechanism resembling but different fro m P-gp can modulate steroid entry into these mouse fibroblasts. This i s confirmed by the failure to demonstrate the presence of P-gp by immu noprecipitation and Western blot experiments in membrane preparations from both cell lines. From these data, we conclude: (i) that the two s ynthetic GR ligands do not accumulate similarly in mouse fibroblasts, (11) that RU 49 953 increases steroid efflux, in contrast to other age nts known to reverse P-gp activity (iii) that cellular entry and expor t of Dex and TA can be modulated by membrane efflux mechanism(s), diff erent 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-induc ed transcription of all the drugs tested, although they all increase s teroid accumulation, suggests involvement of immunosuppressant-influen ced factor(s) acting downstream from steroid entry, in the hormone rec eptor-mediated transcription pathway(s).