Transcriptional regulation of the murine multidrug resistance gene mdr1b by progesterone occurs via an indirect mechanism.

Abstract

The murine multidrug resistance gene mdr1b is highly induced in the endometrium during pregnancy. Evidence suggests that induction occurs mainly as a result of progesterone action. To study the molecular mechanisms involved in this induction, 5'-flanking sequences between -540 and +97 of the mdr1b gene were fused to the reporter gene, bacterial chloramphenicol acetyltransferase (p540CAT). Unlike most progesterone-responsive genes, mdr1b is preferentially activated by the A form of the progesterone receptor. We now report that activation is not observed with a DNA-binding domain mutant of progesterone receptor A (PRA) suggesting that induction occurs at the transcriptional level. Time course experiments demonstrated that induction was first observed 12 hr after hormone addition, suggestive of a secondary (or late) response gene. Sequence comparison highlighted the region M1 (-234 to -206), which contains a partially conserved progesterone response element. Its functional significance was evaluated by expression assays and gel shift analysis. Reporter plasmids with modifications of this element were transfected into HeLa cells. Constructs containing the native M1 element, or a mutated element (M1mt) that eliminated any similarity to a progesterone response element, were induced four-fold by progesterone whereas an element containing a consensus progesterone response element (M1PRE) was induced eight-fold. In addition, by gel shift analysis, the M1 element did not bind the progesterone receptor or any other factors. This suggested that the M1 region does not participate in the response to progesterone. 5' Nested deletion analysis, used to identify other regions of the upstream regulatory region that contributed to induction by progesterone, demonstrated that enhancer sequences between -122 and -65, which contain binding sites for C/EBPbeta and NF-Y, were important. Mutations in the binding sites for these factors decreased induction by progesterone. On the basis of our studies using 540 bp of upstream sequence, mdr1b is activated transcriptionally by progesterone, in an indirect manner dependent on basal factors.