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.

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).

Calcium/calmodulin kinase inhibitors and immunosuppressant macrolides rapamycin and FK506 inhibit progestin- and glucocorticosteroid receptor-mediated transcription in human breast cancer T47D cells.

The effects of immunosuppressants and inhibitors of specific calcium/calmodulin kinase (CaMK) of types II and IV on progestin/glucocorticosteroid-induced transcription were studied in two human stably transfected breast cancer T47D cell lines. The lines contain the chloramphenicol acetyl transferase (CAT) gene under control either of the mouse mammary tumor virus promoter (T47D-MMTV-CAT), or the minimal promoter containing five glucocorticosteroid/progestin hormone response elements [T47D-(GRE)5-CAT]. Progestin- and triamcinolone acetonide (TA)-induced CAT gene expression was inhibited in a dose-dependent manner in both lines by preincubation with rapamycin (Rap) and, to a lesser extent, with FK506, but not with cyclosporin A. CaMK II and/or IV inhibitors KN62 and KN93 also inhibited progestin- and TA-stimulated transcription in both lines. None of these drugs had any effect on basal transcription. The antagonist RU486 inhibited all the effects of both progestin and TA, suggesting that progesterone receptor (PR)-, as well as glucocorticosteroid receptor (GR)- mediated transactivation are targets of immunosuppressants and CaMKs in T47D cells. Indeed, Northern analysis showed that Rap, KN62, and, to a lesser degree, FK506 inhibited progestin stimulation of Cyclin D1 mRNA levels, but not those of the non-steroid-regulated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Addition of Rap or KN62 after exposure of cells to progesterone agonist Org 2058 had no effect on induction of CAT activity. Taken together, these data indicate that Rap and FK506, as well as CaMK inhibitors, inhibit steroid-induced activities of exogenous, as well as of some endogenous, steroid receptor-regulated genes by a mechanism preceding hormone-induced receptor activation. Rap appeared to stabilize a 9S form of [3H]Org 2058-PR complexes isolated from T47D (GRE)5CAT cell nuclei. By contrast, the progesterone receptor (PR) was isolated from cells treated with KN62 as a 5S entity, undistinguishable from the 5S PR species extracted from cells treated with progestin only. The nuclear 9S-[3H]Org2058-PR resulting from cells exposed to Rap, contained, in addition to the heat shock proteins of 90 kDa and 70 kDa (hsp90 and hsp70), the FK506-binding immunophilin FKBP52 but not FKBP51, although the latter was part of unliganded PR heterocomplex associated with hsp90. These results suggest that Rap and KN62 act upon the PR by distinct mechanisms, with only Rap impeding progestin-induced PR transformation. FKBP51 appeared to dissociate from the receptor heterocomplex, but not from hsp90, after hormone binding to PR in vitro and in vivo, whether in the presence or not of Rap and KN62. Immunoprecipitation experiments distinguished two PR- and glucocorticosteroid (GR)-associated molecular chaperone complexes, containing hsp90 and hsp70 and FKBP52 or FKBP51. Another complex identified in T47D cytosol contained hsp90 and the cyclosporin A-binding cyclophilin of 40 kDa, CYP40, but not hsp70, PR, or GR. These observations support the concept that FKBP51 and FKBP52 can act as regulators of Rap and FK506 activity upon PR and GR-mediated transcription, a mechanism that could be also regulated by type II and/or type IV CaMKs.

Cyclosporin A potentiates the dexamethasone-induced mouse mammary tumor virus-chloramphenicol acetyltransferase activity in LMCAT cells: a possible role for different heat shock protein-binding immunophilins in glucocorticosteroid receptor-mediated gene expression.

As previously observed for FK506, we report here that cyclosporin A (CsA) treatment of mouse fibroblast cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid (LMCAT cells) results in potentiation of dexamethasone (Dex)-induced CAT gene expression. Potentiation by CsA is observed in cells treated with 10-100 nM Dex but not in cells treated with 1 microM Dex, a concentration of hormone which results in maximum CAT activity. At 10 nM Dex, 1-5 microM CsA provokes an approximately 50-fold increase in CAT gene transcription, compared with transcription induced by Dex alone. No induction of CAT gene expression is observed in cells treated with CsA or FK506 in the absence of Dex. The antisteroid RU 486 abolishes effects obtained in the presence of Dex. Using a series of CsA, as well as FK506, analogs, including some devoid of calcineurin phosphatase inhibition activity, we conclude that the potentiation effects of these drugs on Dex-induced CAT gene expression in LMCAT cells do not occur through a calcineurin-mediated pathway. Western-blotting experiments following immunoprecipitation of glucocorticosteroid receptor (GR) complexes resulted in coprecipitation of GR, heat shock protein hsp90 and two immunophilins: the FK506-binding protein FKBP59 and the CsA-binding protein cyclophilin 40 (CYP40). Two separate immunophilin-hsp90 complexes are present in LMCAT cells: one containing CYP40-hsp90, the other FKBP59-hsp90. Thus, both FKBP59 and CYP40 can be classified as hsp-binding immunophilins, and their possible involvement as targets of immunosuppressants potentiating the GR-mediated transcriptional activity is discussed.

Effects of immunosuppressants FK506 and rapamycin on the heterooligomeric form of the progesterone receptor.

The non-DNA binding form of the rabbit uterus cytosol progesterone receptor (PR) contains, in addition to the hormone binding unit and heat shock protein M(r) 90kDa (hsp90), a Heat shock protein Binding Immunophilin (p59/HBI) which interacts with hsp90. P59/HBI binds the immunosuppressants FK506 and Rapamycin (RAP) and belongs to the FK506 binding protein family. A recombinant p59/HBI-glutathione-S-transferase fusion protein, purified by Sephadex LH-20 filtration of tritiated drug-p59/HBI complexes, binds FK506 and RAP with apparent Kd values of 75 +/- 40 and 40 +/- 15 nM, respectively. Immunopurification from cytosol of [3H]steroid-labeled tungstate-stabilized PR with anti-PR immunoadsorbent yielded "9S"-PR species in which hsp90, hsp70 and p59/HBI were present. In the absence of tungstate ions, only the 4-6S PR was eluted, and Western blot analysis demonstrated the absence of hsps and p59/HBI. In contrast 30 to 50% of the original 9S-PR species containing hsps and p59/HBI, was eluted in the absence of tungstate ions but after exposure of cytosol to 5 microM FK506 or RAP. Other experiments showed that cytosol fractions incubated for 2 h at 25 degrees C with 0.05 to 10 microM FK506 or RAP, then with [3H]steroids (the agonist [3H]Org 2058 or the anti-progestin [3H]RU486), contains greater amounts of 9S-PR species than that detected in non-immunosuppressant exposed control cytosol. Scatchard analysis showed an up to 2-fold decrease of the Kd value for both hormones following exposure to drugs, without modification of the number of steroid binding sites. Purification of cytosol PR on immobilized FK506 yields a 9S form still containing hsp90, hsp70 and p59/HBI associated to PR units. Altogether, these results suggest that binding of immunosuppressants to p59/HBI does not promote hsps dissociation from the receptor and, as a consequence, that inhibition of peptidyl-prolyl isomerase activity of p59/HBI by immunosuppressants binding does not transform (activate) PR in vitro. However, given the assumption that hsp90 binds to receptor and that p59/HBI binds hsp90 but not directly to receptor, immunosuppressants affect hormone binding by an unknown mechanism involving receptor associated proteins. In addition, we show that the chick oviduct cytosol 9S-PR, not displaced with the EC1 antibody specific for several mammalian p59/HBI, also binds to FK506 columns and can be eluted by exchange with either FK506 or RAP, suggesting that there is an avian HBI homolog.(ABSTRACT TRUNCATED AT 400 WORDS)

The mammalian heat shock protein binding immunophilin (p59/HBI) is an ATP and GTP binding protein.

A protein of M(r) 59,000 (p59) was recently cloned and identified as a Heat shock protein Binding Immunophilin (p59/HBI). It participates to the heterooligomeric, non-DNA binding form of steroid receptors, in association with the heat shock protein of M(r) 90,000 (hsp90). It binds the immunosuppressants FK506 and rapamycin and possesses three FKBP-12 (FK506 binding protein of M(r) 12,000)--like domains (I to III), plus a tail containing a putative calmodulin binding site (domain IV). Following expression in E. Coli and purification on Glutathione-Sepharose of either the full-length recombinant p59/HBI, or the recombinant FKBP-like domains, we demonstrate by autoradiography of [alpha 32P]-8-azido ATP and of [alpha 32P]-8-azido GTP photoaffinity labeled complexes, that an ATP (GTP) binding site is located in the domain II. This nucleotide binding property is also found with the highly purified rabbit uterus p59/HBI. The latter, but not the recombinant protein, can be phosphorylated in vitro in the presence of Mn++ and/or of Ca++/Calmodulin in an ATP but not GTP dependent manner, suggesting copurification of a CaM kinase II-like enzyme. Thus it appears that p59/HBI is a multifunctional immunophilin which may be at the crossroad of the endocrine and immunological systems.

[Aspects of the tilted disk syndrome]. / Les pièges de la dysversion papillaire.

4 cases of tilted disc syndrome are reported. Two were associated with sellar or endocrine pathology, one had a particular aspect of chorioretinal atrophy, and the last had a thrombosis of the central vein of retina. The most commonly aspects of tilted disc syndrome are reported.