Functional analysis of the murinemdr1b promoter and its regulation by progesterone

Abstract

Multidrug resistance is characterized by the cross resistance of cells to antitumor agents that are structurally and functionally unrelated. This resistance is mediated by a membrane protein, P-glycoprotein (P-gp), that is involved in the active transport of antitumor agents from cells. P-gp, the product of the mdr multigene family, is expressed under normal physiological conditions in several tissues including the liver, kidney, adrenal gland and intestine and is highly expressed during pregnancy on the lumenal surface of the secretory epithelium in the gravid uterus of mice, rats and hamsters. In rodents there are three genes; mdr1a, mdr1b and mdr2 whereas in humans there are two genes. It is the mdr1b gene product only that is expressed in the gravid uterus of rodents. In the human endometrium, P-gp is also expressed during pregnancy and in the secretory phase of the menstrual cycle. The 5{dollar}\sp\prime{dollar} flanking sequences of the mdr1b gene were isolated, subcloned and sequenced, and their promoter activity was analyzed. An expression vector, mdr1b-CAT, was constructed by fusion of this promoter region to a reporter gene, the bacterial chloramphenicol acetyltransferase (CAT) gene. A series of 5{dollar}\sp\prime{dollar} deleted clones of the mdr1b-CAT construct identified several regions that appear to play a role in promoter activity. R5020, a progesterone agonist, increased the expression of mdr1b-CAT when transfected into T47D cells, a line that constitutively expresses the progesterone receptor. Moreover, a greater response to R5020 was observed when the cells were co-transfected with an expression vector for the A form of the progesterone receptor, but not the B form. A series of 5{dollar}\sp\prime{dollar} deleted clones of the mdr1b-CAT construct indicated that the region of responsiveness was located in the first untranslated exon of the gene. Furthermore, sequences from the first exon were able to confer responsiveness to the non-responsive TK-CAT vector. This study demonstrates that progesterone specifically regulates the activity of the mdr1b promoter and that this response is directed solely by the A form of the progesterone receptor.