No abstract

The binding of mineralocorticoid hormones to the mineralocorticoid receptor is the first step in a cascade of events leading to the stimulation of Na ϩ reabsorption by renal cortical collecting duct (CCD) principal cells. The agonist properties of mineralocorticoid hormones are linked to contacts between their 21-hydroxyl group and Asn770, a residue of the ligand-binding domain of the human mineralocorticoid receptor (hMR). Here, we investigate whether the presence of a hydroxyl group at position 11, 17, or 20 could also alter the activity of progesterone (P), a mineralocorticoid antagonist without the 21-hydroxyl group. Both 17␣-hydroxyprogesterone (17OHP) and 20␣-hydroxyprogesterone (20OHP) antagonized the aldosterone-induced trans-activation activity (IC 50 : 17OHP, 10 Ϫ7 M; 20OHP, 10 Ϫ8 M) of the hMR transiently expressed in COS-7 cells lacking steroid receptors. In cultured mouse mpkCCD cl4 principal cells, 17OHP and 20OHP also prevented the aldosterone-stimulated amiloride-sensitive component of the short-circuit current (Ams I sc ), reflecting Na ϩ absorption mediated by the epithelial Na ϩ channel (ENaC). In contrast, 11␤-hydroxyprogesterone (11OHP) activated the transiently expressed hMR in COS-7 cells in a dose-dependent manner (ED 50 : 10 Ϫ8 M) and, like aldosterone, stimulated Ams I sc in mpkCCD cl4 cells. Docking 11OHP within the hMR-ligand-binding domain homology model revealed that the agonist activity of 11OHP is caused by contacts between its 11␤-hydroxyl group and Asn770. Furthermore, 11OHP was unable to activate the mutant hMR/N770A, in which Ala is substituted for Asn at position 770. These findings demonstrate that in the absence of the 21-hydroxyl group, the 11␤-hydroxyl group can produce the contact with the hMRAsn770 required for the hMR activation leading to stimulated Na ϩ absorption.In the kidney, the collecting duct is the main site of Na ϩ reabsorption and is subjected to a fine hormonal control by aldosterone and vasopressin (Rossier and Palmer, 1992). In the principal cells of the cortical collecting duct (CCD), sodium enters via the amiloride-sensitive epithelial sodium channel (ENaC) and exits via the basolaterally located Na,KATPase pump. The regulation of sodium reabsorption by aldosterone requires it to be bound to the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily (Arriza et al., 1987). These receptors share a common modular structure with three major functional domains. The N-terminal region contains a constitutive trans-activation function. The central DNA-binding domain consists of two zinc fingers that are involved in DNA binding and receptor dimerization. The ligand-binding domain (LBD) lies in the C-terminal region and is involved in several functions, including nuclear localization, interaction with the 90-kDa heat-shock protein, homo-and/or hetero-dimerization, and a ligand-dependent activation function (Evans, 1988;Tsai and O'Malley, 1994;Mangelsdorf et al., 1995;Ribeiro et al., 1995).The crystal structure of the MR-LBD has not ...

Nucleic acid aptamers are often referred to as chemical antibodies. Because they possess several advantages, like their smaller size, temperature stability, ease of chemical modification, lack of immunogenicity and toxicity, and lower cost of production, aptamers are promising tools for clinical applications. Aptamers against cell surface protein biomarkers are of particular interest for cancer diagnosis and targeted therapy. In this study, we identified and characterized RNA aptamers targeting cells expressing integrin α5β1. This αβ heterodimeric cell surface receptor is implicated in tumor angiogenesis and solid tumor aggressiveness. In glioblastoma, integrin α5β1 expression is associated with an aggressive phenotype and a decrease in patient survival. We used a complex and original hybrid SELEX (selective evolution of ligands by exponential enrichment) strategy combining protein-SELEX cycles on the recombinant α5β1 protein, surrounded by cell-SELEX cycles using two different cell lines. We identified aptamer H02, able to differentiate, in cyto- and histofluorescence assays, glioblastoma cell lines, and tissues from patient-derived tumor xenografts according to their α5 expression levels. Aptamer H02 is therefore an interesting tool for glioblastoma tumor characterization.