CALCIUM CALMODULIN KINASE INHIBITORS AND IMMUNOSUPPRESSANT MACROLIDESRAPAMYCIN AND FK506 INHIBIT PROGESTIN-MEDIATED AND GLUCOCORTICOSTEROID RECEPTOR-MEDIATED TRANSCRIPTION IN HUMAN BREAST-CANCER T47D CELLS/

The effects of immunosuppressants and inhibitors of specific calcium/c almodulin kinase (CaMK) of types II and IV on progestin/glucocorticost eroid-induced transcription were studied in two human stably transfect ed breast cancer T47D cell lines. The lines contain the chloramphenico l acetyl transferase (CAT) gene under control either of the mouse mamm ary tumor virus promoter (T47D-MMTV-CAT), or the minimal promoter cont aining five glucocorticosteroid/progestin hormone response elements [T 47D-(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, w ith FK506, but not with cyclosporin A. CaMK II and/or IV inhibitors KN 62 and KN93 also inhibited progestin- and TA-stimulated transcription in both lines. None of these drugs had any effect on basal transcripti on. The antagonist RU486 inhibited all the effects of both progestin a nd TA, suggesting that progesterone receptor (PR)-, as well as glucoco rticosteroid receptor (GR)- mediated transactivation are targets of im munosuppressants and CaMKs in T47D cells. Indeed, Northern analysis sh owed that Rap, KN62, and, to a lesser degree, FK506 inhibited progesti n stimulation of Cyclin D1 mRNA levels, but not those of the non-stero id-regulated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Ad dition of Rap or KN62 after exposure of cells to progesterone agonist Org 2058 had no effect on induction of CAT activity. Taken together, t hese data indicate that Rap and FK506, as well as CaMK inhibitors, inh ibit steroid-induced activities of exogenous, as well as of some endog enous, steroid receptor-regulated genes by a mechanism preceding hormo ne-induced receptor activation. Rap appeared to stabilize a 9S form of [H-3]Org 2058-PR complexes isolated from T47D (GRE)(5)CAT cell nuclei . By contrast, the progesterone receptor (PR) was isolated from cells treated with KN62 as a 5S entity, undistinguishable from the 5S PR spe cies extracted from cells treated with progestin only. The nuclear 9S- [H-3]Org2058-PR resulting from cells exposed to Rap, contained, in add ition 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 me chanisms, 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 experimen ts distinguished two PR- and glucocorticosteroid (GR)associated molecu lar chaperone complexes, containing hsp90 and hsp70 and FKBP52 or FKBP 51. 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 OR-mediat ed transcription, a mechanism that could be also regulated by type II and/or type IV CaMKs.