Tissue-specific regulation of the ecto-5'-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region.

We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.     

Localization of breast cancer resistance protein (Bcrp) in endocrine organs and inhibition of its transport activity by steroid hormones

Breast cancer resistance protein (BCRP) is known for its protective function against the toxic effects of exogenous compounds. In addition to this, a role in the transport of endogenous compounds has been described. Since BCRP in the plasma membrane was shown to be regulated by sex steroids, we investigated the presence and possible role of BCRP in steroid hormone-producing organs. Therefore, the presence and localization of Bcrp was investigated in endocrine organs of wild-type mice. Furthermore, the interaction of various steroid hormones with human BCRP activity was studied. Quantitative PCR revealed Bcrp mRNA in the pituitary and adrenal glands, pancreas, ovary, testis and adipose tissue. Immunohistochemistry revealed the presence of Bcrp in the cortex of the adrenal gland and in plasma membranes of adipocytes. In the pituitary gland, pancreas, ovary and testis, Bcrp was mainly located in the capillaries. The interaction between BCRP and 12 steroid hormones was studied using membrane vesicles of HEK293-BCRP cells. Estradiol, testosterone, progesterone and androstenedione inhibited BCRP-mediated uptake of (3)H-estrone sulphate (E(1)S) most potently, with calculated inhibitory constant (Ki) values of 5.0?±?0.2, 36?±?14, 14.7?±?1.3 and 217?±?13?μM, respectively. BCRP function was attenuated non-competitively, which implies an allosteric inhibition of BCRP-mediated E(1)S transport by these steroids. In conclusion, localization of Bcrp in endocrine organs together with the efficient allosteric inhibition of the efflux pump by steroid hormones are suggestive for a role for BCRP in steroid hormone regulation.     

Tissue distribution and hormonal regulation of the breast cancer resistance protein (Bcrp/Abcg2) in rats and mice

Breast cancer resistance protein (Bcrp/Abcg2) is a member of the ABC transporter family. The purpose of this study was to quantify Bcrp mRNA in rat and mouse tissues, and to determine whether there are gender differences in Bcrp mRNA expression. Rat Bcrp mRNA levels were high in intestine and male kidney, and intermediate in testes. Mouse Bcrp expression was highest in kidney, followed by liver, ileum, and testes. Male-predominant expression of Bcrp was observed in rat kidney and mouse liver. Furthermore, gonadectomy and hypophysectomy experiments were conducted to determine whether sex steroids and/or growth hormone are responsible for Bcrp gender-divergent expression patterns. Male-predominant expression of Bcrp in rat kidney appears to be due to the suppressive effect of estradiol, and male-predominant expression of Bcrp in mouse liver appears to be due to the inductive effect of testosterone.