11β-Hydroxyprogesterone Acts as a Mineralocorticoid Agonist in Stimulating Na⁺Absorption in Mammalian Principal Cortical Collecting Duct Cells

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 ...

Aldosterone Activates NF-κB in the Collecting Duct

“…Luciferase activity weakly increased in response to 10 Ϫ6 M dexamethasone, but this stimulation was 2.5-fold lower than that produced by 10 Ϫ6 M aldosterone ( Figure 8A), suggesting that the MR rather than the GR mediates aldosterone-induced NF-B activation. In agreement with this hypothesis, the progesterone derivative 17-␣-hydroxyprogesterone, that specifically antagonizes MR transactivation without significantly altering GR activity 23 (unpublished data; see the Concise Methods section) also fully prevented the effect of 10 Ϫ6 M aldosterone on NF-B-dependent gene transcription ( Figure 8B). Moreover, siRNA targeting the MR, which decreased MR abundance by 70% without altering GR expression ( Figure 8C), prevented aldosterone-induced IB␣, IL-6, and PAI-1 mRNA expression ( Figure 8D), whereas effective knockdown of the GR did not ( Figure 9, A and B).…”

“…In all experiments using one or more of these compounds, control cells were incubated in the presence of the same concentration of DMSO as treated cells. As shown by RafestinOblin et al, 23 17OHP behaves as a competitive inhibitor of the MR and was shown to antagonize up to 80% of aldosterone-induced transactivation activity of the MR. 17OHP also attenuated the aldosteronestimulated amiloride-sensitive short-circuit current in murine mpkCCD cl4 cells. In addition, 17OHP is a weak agonist/antagonist of the GR because the GR transactivation activity induced by 10 Ϫ6 M 17OHP amounted to approximately 15% of that induced by 10…”

The Synthetic Androgen Methyltrienolone (R1881) Acts as a Potent Antagonist of the Mineralocorticoid Receptor

“…This is the case, for instance, of progesterone, the antagonist activity of which has been linked to its inability to establish contact with the Asn770 residue of MR-LBD . Two progesterone derivatives, 17␣-and 20␣-hydroxyprogesterone (but not 11␤-hydroxyprogesterone) act as potent MR antagonists and inhibit the aldosterone-stimulated absorption of Na ϩ in cultured mouse mpkCCD cl4 collecting duct principal cells (Rafestin-Oblin et al, 2002). Rossier et al (1980) first reported that testosterone (T) specifically displaces [ 3 H]aldosterone from its nuclear and cytoplasmic binding sites and antagonizes the stimulating action of aldosterone on Na ϩ transport in the toad bladder.…”

“…In this study, the ability of the two main natural androgens, T and its active metabolite dihydrotestosterone (DHT), and the potent synthetic steroidal AR agonist methyltrienolone (R1881) to modulate Na ϩ absorption was analyzed using the short-circuit current (I sc ) method in cultured mpkCCD cl4 collecting duct cells, which had retained the main features of intact CCDs, including a Na ϩ transport mechanism that was stimulated by aldosterone (Bens et al, 1999;Rafestin-Oblin et al, 2002). The ability of T, DHT, and R1881 to activate or inactivate MR was investigated in cis-trans cotransfection assays in HEK 293T and COS-7 cells.…”

Drug Design Approaches to Manipulate the Agonist-Antagonist Equilibrium in Steroid Receptors

“…Thr739 is the last residue in helix-11 whose mutation to alanine has no effect on the binding of triamcinolone acetonide, but does result in a 16-fold reduction in transactivation (Lind et al, 2000). In addition to these mutation studies, as discussed already, there is also overwhelming evidence across the family to support the hypothesis that Asn564 is crucial for the agonistic activity of GR and related receptors (Bledsoe et al, 2005;Bledsoe, Stewart, and Pearce, 2004;Fagart et al, 1998;Hellal-Levy et al, 2000;Necela and Cidlowski, 2003;Rafestin-Oblin et al, 2002). The role of Asn564 (Asn705 in AR, Asn770 in MR) was previously discussed.…”