Studies on the regulation of the murine multidrug resistance genemdr1b by progesterone

Abstract

The murine multidrug resistance gene mdr1b is highly induced in the endometrium during pregnancy. Induction occurs mainly as a result of progesterone action. Progesterone belongs to the family of steroid hormones whose receptors function as ligand-activated transcription factors that bind to their cognate response elements in the regulatory regions of many genes. 5{dollar}\sp\prime{dollar} flanking sequences between {dollar}-{dollar}540 and +97 of the mdr1b gene were fused to the reporter gene bacterial chloramphenicol acetyltransferase (p540CAT). In transient transfections, p540CAT was induced upon treatment with R5020, a progesterone analog, when cotransfected with the A form (PR{dollar}\sb{lcub}\rm A{rcub}{dollar}), but not the B form (PR{dollar}\sb{lcub}\rm B{rcub}{dollar}) of the receptor. p540CAT cotransfected with a DNA binding domain mutant of PR{dollar}\sb{lcub}\rm B{rcub}{dollar} was not induced. Coexpression of PR{dollar}\sb{lcub}\rm A{rcub}{dollar} and PR{dollar}\sb{lcub}\rm B{rcub}{dollar} inhibited the induction observed with PR{dollar}\sb{lcub}\rm A{rcub}{dollar} alone.;To determine whether mdr1b was an early or a late response gene for progesterone, a time course of activation was performed in HeLa cells transiently cotransfected with p540CAT and an expression vector for PR{dollar}\sb{lcub}\rm A{rcub}{dollar}. Activation was observed 12 hours after hormone addition and peaked at 18 hours suggestive of a late gene. Sequences between {dollar}-{dollar}234 and {dollar}-{dollar}206 which contain a partially conserved progesterone response element (mdrPRE) were modified to create a consensus PRE (cPRE) or mutated, to eliminate any similarity to a PRE (mPRE). In HeLa cells cotransfected with PR{dollar}\sb{lcub}\rm A{rcub}{dollar}, mPRE or wildtype were induced 4-fold while cPRE induced expression 8-fold. It suggests that mdrPRE, although partially conserved, does not function as a progesterone response element in this context. By gel shift analysis, the region containing mdrPRE does not bind any nuclear factors. Taken together, the mdr1b gene appears to be a late gene activated by indirect mechanisms.;Nested deletion analysis to identify sequences required for activation by progesterone was performed. Regions were designated A-E. Two regions D and E were further studied. Region D between {dollar}-{dollar}122 and {dollar}-{dollar}65 has previously been shown to bind C/EBP{dollar}\beta{dollar} and NF-Y and mutation of these binding sites decreased induction by progesterone. Region E contained sequences from {dollar}-{dollar}65 and including the first untranslated exon made a modest contribution to the overall progesterone response. In summary, mdr1b is a late gene and induction by progesterone occurs via a complex regulatory mechanism that depends on the factors C/EB{dollar}\beta{dollar} and NF-Y.