RESUMO
Aldosterone regulates sodium homeostasis by activating the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Hyperaldosteronism leads todeleterious effects on the kidney, blood vessels, and heart. Although steroidal antagonists such as spironolactone and eplerenone are clinically useful for the treatment of cardiovascular diseases, they are associated with several side effects. Finerenone, a novel nonsteroidal MR antagonist, is presently being evaluated in two clinical phase IIb trials. Here, we characterized the molecular mechanisms of action of finerenone and spironolactone at several key steps of the MR signaling pathway. Molecular modeling and mutagenesis approaches allowed identification of Ser-810 and Ala-773 as key residues for the high MR selectivity of finerenone. Moreover, we showed that, in contrast to spironolactone, which activates the S810L mutant MR responsible for a severe form of early onset hypertension, finerenone displays strict antagonistic properties. Aldosterone-dependent phosphorylation and degradation of MR are inhibited by both finerenone and spironolactone. However, automated quantification of MR subcellular distribution demonstrated that finerenone delays aldosterone-induced nuclear accumulation of MR more efficiently than spironolactone. Finally, chromatin immunoprecipitation assays revealed that, as opposed to spironolactone, finerenone inhibits MR, steroid receptor coactivator-1, and RNA polymerase II binding at the regulatory sequence of the SCNN1A gene and also remarkably reduces basal MR and steroid receptor coactivator-1 recruitment, unraveling a specific and unrecognized inactivating mechanism on MR signaling. Overall, our data demonstrate that the highly potent and selective MR antagonist finerenone specifically impairs several critical steps of the MR signaling pathway and therefore represents a promising new generation MR antagonist.
Assuntos
Aldosterona/farmacologia , Naftiridinas/farmacologia , Coativador 1 de Receptor Nuclear/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Western Blotting , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Células HEK293 , Humanos , Cinética , Microscopia de Fluorescência , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Receptores de Mineralocorticoides/genética , Transdução de Sinais/efeitos dos fármacos , Espironolactona/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
There is a therapeutic need for glucocorticoid receptor (GR) ligands that distinguish between the transrepression and transactivation activity of the GR, the later thought to be responsible for side effects. These ligands are known as "dissociated glucocorticoids" (dGCs). The first published dGCs, RU24782 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-thiomethyl-1,4-diene-3,20-dione) and RU24858 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-cyanide-1,4-diene-3,20-dione), do not have the 17α-hydroxyl group that characterizes dexamethasone (Dex; 9α-fluoro-11ß,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione), and they differ from one another by having C21-thiomethyl and C21-cyanide moieties, respectively. Our aim was therefore to establish the structural basis of their activity. Both RU24782 and RU24858 induced a transactivation activity highly dependent on the GR expression level but always lower than dexamethasone. They also display less ability than dexamethasone to trigger steroid receptor coactivator 1 (SRC-1) recruitment and histone H3 acetylation. Docking studies, validated by mutagenesis experiments, revealed that dGCs are not anchored by Gln642, in contrast to Dex, which is hydrogen bonded to this residue via its 17α-hydroxyl group. This contact is essential for SRC-1 recruitment and subsequent dexamethasone-induced GR transactivation, but not transrepression. The ability of dGCs to make contacts with Ile747, for both RU24858 and RU24782 and with Asn564 for RU24858 are not strong enough to maintain GR in a conformation able to efficiently recruit SRC-1, unless SRC-1 is overexpressed. Overall, our findings provide some structural guidelines for the synthesis of potential new dissociated glucocorticoids with a better therapeutic ratio.
Assuntos
Glucocorticoides/farmacologia , Receptores de Glucocorticoides/genética , Ativação Transcricional/efeitos dos fármacos , Transporte Ativo do Núcleo Celular , Animais , Sítios de Ligação , Células COS , Células Cultivadas , Chlorocebus aethiops , Dexametasona/farmacologia , Glucocorticoides/química , Glucocorticoides/metabolismo , Humanos , Coativador 1 de Receptor Nuclear/fisiologia , Regiões Promotoras Genéticas , Conformação Proteica , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/genéticaRESUMO
Pseudohypoaldosteronism type 1 (PHA1) is a rare genetic disease of mineralocorticoid resistance characterized by salt wasting and failure to thrive in infancy. Here we describe the first case of a newborn with severe recessive PHA1 caused by two heterozygous mutations in NR3C2, gene coding for the mineralocorticoid receptor (MR). Independent segregation of the mutations occurred in the family, with p.Ser166X being transmitted from the affected father and p.Trp806X from the asymptomatic mother Whereas the truncated MR(166X) protein was degraded, MR(806X) was expressed both at the mRNA and protein level. Functional studies demonstrated that despite its inability to bind aldosterone, MR(806X) had partial ligand-independent transcriptional activity. Partial nuclear localization of MR(806X) in the absence of hormone was identified as a prerequisite to initiate transcription. This exceptional case broadens the spectrum of clinical phenotypes of PHA1 and demonstrates that minimal residual activity of MR is compatible with life. It also suggests that rare hypomorphic NR3C2 alleles may be more common than expected from the prevalence of detected PHA1 cases. This might prove relevant for patient's care in neonatal salt losing disorders and may affect renal salt handling and blood pressure in the general population.
Assuntos
Códon sem Sentido/genética , Insuficiência de Crescimento/genética , Hiponatremia/genética , Pseudo-Hipoaldosteronismo/genética , Receptores de Mineralocorticoides/genética , Aldosterona/metabolismo , Animais , Células COS , Pré-Escolar , Chlorocebus aethiops , Códon de Terminação/genética , Saúde da Família , Feminino , Humanos , Recém-Nascido , Masculino , Linhagem , Ligação Proteica/genética , Receptores de Mineralocorticoides/metabolismo , Índice de Gravidade de DoençaRESUMO
Limitations of current steroidal mineralocorticoid receptor (MR) antagonists have stimulated the search for a new generation of molecules. We screened for novel nonsteroidal compounds and identified MR antagonists derived from the chemical class of dihydropyridines. Chemical optimization resulted in BR-4628, which displays high in vitro and in vivo MR potency as well as selectivity with respect to the other steroid hormone receptors and the L-type calcium channel. Biochemical studies demonstrated that BR-4628 forms complexes with MR that do not promote the recruitment of transcriptional co-regulators. Docking experiments, using the crystal structure of the MR ligand-binding domain in an agonist conformation, revealed that BR-4628 accommodates in the MR ligand-binding cavity differently in comparison with the classical steroidal MR antagonists. An alanine scanning mutagenesis approach, based on BR-4628 docking, allowed identifying its anchoring mode within the ligand-binding cavity. Altogether, we propose that BR-4628 is a bulky antagonist that inactivates MR through a passive mechanism. It represents the prototype of a new class of MR antagonists.
Assuntos
Di-Hidropiridinas/farmacologia , Antagonistas de Receptores de Mineralocorticoides , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Cricetinae , Cricetulus , Cristalografia por Raios X , Di-Hidropiridinas/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Mutação de Sentido Incorreto , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismoRESUMO
Many progestins have been developed for use in contraception, menopausal hormone therapy, and treatment of gynecological diseases. They are derived from either progesterone or testosterone, and they act by binding to the progesterone receptor (PR), a hormone-inducible transcription factor belonging to the nuclear receptor superfamily. Unlike mineralocorticoid, glucocorticoid, and androgen receptors, the steroid-receptor contacts that trigger the switch of the ligand-binding domain from an inactive to an active conformation have not yet been identified for the PR. With this aim, we solved the crystal structure of the ligand-binding domain of the human PR complexed with levonorgestrel, a potent testosterone-derived progestin characterized by a 13-ethyl substituent. Via mutagenesis analysis and functional studies, we identified Met909 of the helix 12 as the key residue for PR activation by both testosterone- and progesterone-derived progestins with a 13-methyl or a 13-ethyl substituent. We also showed that Asn719 contributes to PR activation by testosterone-derived progestins only, and that Met759 and Met909 are responsible for the high potency of 19-norprogestins and of 13-ethyl progestins, respectively. Our findings provide a structural guideline for the rational synthesis of potent PR agonist and antagonist ligands that could have therapeutic uses in women's health.
Assuntos
Congêneres da Progesterona/farmacologia , Progestinas/farmacologia , Receptores de Progesterona/agonistas , Linhagem Celular , Cristalização , Cristalografia por Raios X , Humanos , Levanogestrel/química , Modelos Moleculares , Mutação , Congêneres da Progesterona/química , Progestinas/química , Conformação Proteica , Ensaio Radioligante , Receptores de Progesterona/química , Receptores de Progesterona/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-Atividade , Transcrição Gênica , Ativação TranscricionalRESUMO
The mineralocorticoid receptor (MR) binds aldosterone, but also glucocorticoid hormones (corticosterone in rodents, cortisol in humans), which largely prevail in the plasma. To prevent permanent and maximal occupancy of MR by glucocorticoid hormones in aldosterone-target cells, specific effects of aldosterone require metabolism of glucocorticoid hormones into 11-dehydroderivatives by 11-beta hydroxysteroid dehydrogenase (11-HSD2). We analyzed the effect of corticosterone or 11-dehydrocorticosterone (11-DHC) on the transactivation activity of the MR, transiently expressed in a new renal cell line expressing 11-HSD2. We show that, because of its metabolism by 11-HSD2, corticosterone is a poor activator of MR transactivation, except at micromolar concentrations, where the enzyme is saturated. We also show that high micromolar concentrations of 11 DHC are required to activate the MR. The weak antagonist property of 11-DHC on aldosterone-induced hMR transactivations is also documented. Such partial agonist activity of 11-DHC is discussed in the light of its positioning in a three-dimensional model of the MR ligand-binding domain.
Assuntos
Corticosterona/análogos & derivados , Glucocorticoides/metabolismo , Hidroxiesteroide Desidrogenases/metabolismo , Receptores de Mineralocorticoides/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2 , Animais , Western Blotting , Linhagem Celular , Corticosterona/metabolismo , Relação Dose-Resposta a Droga , Humanos , Rim/citologia , Ligantes , Microscopia de Fluorescência , Modelos Moleculares , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Fatores de Tempo , Ativação Transcricional , TransfecçãoRESUMO
The gamma subunit of Na/K/ATPase is a small membrane protein that shares homologies with other members of the FXYD family, like phospholemman and CHIF (corticosteroid hormone-induced factor). Both the gamma subunit and CHIF modulate sodium pump properties. The gamma subunit increases the apparent affinity of the pump for ATP and reduces its apparent affinity for sodium. CHIF, in contrast, augments its apparent affinity for sodium. Gamma subunit expression is essentially restricted to the kidney, with two main splice variants, gammaa and gammab, which differ only at their extracellular N-termini. We have investigated in detail the cell-specific expression of the two splice variants of gamma within the kidney and compared it to that of CHIF. While both gamma variants affect catalytic properties of the pump (without detectable difference between a and b forms), their localization along the nephron is partially distinct. Both variants are coexpressed in the proximal tubule and in the medullary part of the thick ascending limb of Henle's loop (TAL). In contrast, their expression differs in the downstream tubular segments. Within the renal cortex, the sole gamma a variant was found in macula densa cells and in principal cells of the initial parts of the collecting duct. Gamma b is in the cortical part of the TAL. Outer and inner medullary collecting ducts lack detectable gamma expression. These latter nephron segments express CHIF, and no overlap between gamma and CHIF expression along the nephron was observed. Such distinct cell-specific expression argues for complementary roles to modulate Na/K/ATPase activity.
Assuntos
Túbulos Renais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Processamento Alternativo , Animais , Variação Genética , Medula Renal/enzimologia , Medula Renal/metabolismo , Túbulos Renais/enzimologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Currently available progesterone (P4) receptor (PR) antagonists, such as mifepristone (RU486), lack specificity and display partial agonist properties, leading to potential drawbacks in their clinical use. Recent x-ray crystallographic studies have identified key contacts involved in the binding of agonists and antagonists with PR opening the way for a new rational strategy for inactivating PR. We report here the synthesis and characterization of a novel class of PR antagonists (APRn) designed from such studies. The lead molecule, the homosteroid APR19, displays in vivo endometrial anti-P4 activity. APR19 inhibits P4-induced PR recruitment and transactivation from synthetic and endogenous gene promoters. Importantly, it exhibits high PR selectivity with respect to other steroid hormone receptors and is devoid of any partial agonist activity on PR target gene transcription. Two-hybrid and immunostaining experiments reveal that APR19-bound PR is unable to interact with either steroid receptor coactivators 1 and 2 (SRC1 and SCR2) or nuclear receptor corepressor (NcoR) and silencing mediator of retinoid acid and thyroid hormone receptor (SMRT), in contrast to RU486-PR complexes. APR19 also inhibits agonist-induced phosphorylation of serine 294 regulating PR transcriptional activity and turnover kinetics. In silico docking studies based on the crystal structure of the PR ligand-binding domain show that, in contrast to P4, APR19 does not establish stabilizing hydrogen bonds with the ligand-binding cavity, resulting in an unstable ligand-receptor complex. Altogether, these properties highly distinguish APR19 from RU486 and likely its derivatives, suggesting that it belongs to a new class of pure antiprogestins that inactivate PR by a passive mechanism. These specific PR antagonists open new perspectives for long-term hormonal therapy.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Homosteroides/farmacologia , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Receptores de Progesterona/antagonistas & inibidores , Esteroides/farmacologia , Transporte Ativo do Núcleo Celular , Androstenos , Sítios de Ligação , Linhagem Celular Tumoral/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HEK293 , Homosteroides/síntese química , Humanos , Modelos Moleculares , Ligação Proteica , Proteólise/efeitos dos fármacos , Receptores de Progesterona/agonistas , Receptores de Progesterona/metabolismo , Esteroides/síntese química , Fatores de Transcrição/metabolismoRESUMO
The first and critical step in the mechanism of aldosterone action is its binding to the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Over the last 40 years, numerous studies have attempted to determine the structural determinants of ligand-binding to MR. An initial set of data showed that hsp90 is bound to the receptor via specific regions and maintains it in a ligand-binding competent state. Site-directed mutagenesis and functional studies guided by a 3D model of the MR ligand-binding domain (LBD) made it possible to identify the residues responsible for the high affinity and selectivity for aldosterone, and to characterize the mechanisms of MR activation and inactivation. The recent determination of the X-ray crystal structures of the LBD of the wild-type MR and MR(S810L), which is responsible for a familial form of hypertension, has made it possible to elucidate the peculiar mechanism of activation of MR(S810L) and established a clear structure/activity relationship for steroidal and non-steroidal MR antagonists.
Assuntos
Receptores de Mineralocorticoides/química , Receptores de Mineralocorticoides/metabolismo , Aldosterona/química , Aldosterona/metabolismo , Animais , Cristalografia por Raios X , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ligantes , Antagonistas de Receptores de Mineralocorticoides , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptores de Mineralocorticoides/agonistas , Relação Estrutura-AtividadeRESUMO
Spirolactones are potent antagonists of the mineralocorticoid receptor (MR), a ligand-induced transcription factor belonging to the nuclear receptor superfamily. Spirolactones are synthetic molecules characterized by the presence of a C17 gamma-lactone, which is responsible for their antagonist character. They harbor various substituents at several positions of the steroid skeleton that modulate their potency in ways that remain to be determined. This is particularly obvious for C7 substituents. The instability of antagonist-MR complexes makes them difficult to crystallize. We took advantage of the S810L activating mutation in MR (MR(S810L)), which increases the stability of ligand-MR complexes to crystallize the ligand-binding domain (LBD) of MR(S810L) associated with 7alpha-acetylthio-17beta-hydroxy-3-oxopregn-4-en-21-carboxylic acid gamma-lactone (SC9420), a spirolactone with a C7 thioacetyl group. The crystal structure makes it possible to identify the contacts between SC9420 and MR and to elucidate the role of Met852 in the mode of accommodation of the C7 substituent of SC9420. The transactivation activities of MR(S810L/Q776A), MR(S810L/R817A), and MR(S810L/N770A) reveal that the contacts between SC9420 and the Gln776 and Arg817 residues are crucial to maintaining MR(S810L) in its active state, whereas the contact between SC9420 and the Asn770 residue contributes only to the high affinity of SC9420 for MR. Moreover, docking experiments with other C7-substituted spirolactones revealed that the MR(S810L)-activating potency of spirolactones is linked to the ability of their C7 substituent to be accommodated in LBD. It is remarkable that the MR(S810L)-activating and MR(WT)-inactivating potencies of the C7-substituted spirolactones follow the same order, suggesting that the C7 substituent is accommodated in the same way in MR(S810L) and MR(WT). Thus, the MR(S810L) structure may provide a powerful tool for designing new, more effective, MR antagonists.
Assuntos
Antagonistas de Receptores de Mineralocorticoides , Espironolactona/química , Substituição de Aminoácidos , Arginina/genética , Asparagina/genética , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Glicina/genética , Humanos , Ligação de Hidrogênio , Rim/citologia , Ligantes , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Mineralocorticoides/química , Receptores de Mineralocorticoides/genética , Espironolactona/isolamento & purificação , Espironolactona/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo , Ativação Transcricional , TransfecçãoRESUMO
Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15-20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of 125I (a substitute for 36Cl), whereas 22Na passage was unaffected; paracellular permeability to [3H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells.
Assuntos
Aldosterona/fisiologia , Túbulos Renais Coletores/fisiologia , Proteínas de Membrana/metabolismo , Junções Íntimas/fisiologia , Aldosterona/farmacologia , Alcaloides , Animais , Benzofenantridinas , Transporte Biológico Ativo , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Claudina-4 , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Iodetos/metabolismo , Túbulos Renais Coletores/efeitos dos fármacos , Manitol/metabolismo , Ocludina , Fenantridinas/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Ratos , Sódio/metabolismo , Junções Íntimas/efeitos dos fármacosRESUMO
Ubiquitination of proteins such as ion transporters appears to be an important process in the regulation of their membrane expression. Recently, using two-hybrid screening, we have selected a potential partner for the alpha-subunit of the amiloride-sensitive epithelial sodium channel (ENaC): the WWP1 protein, a ubiquitin ligase belonging to the Nedd4 family. To establish whether WWP1 is co-expressed with ENaC, we employed in situ hybridisation, immunohistochemistry and Western blotting to determine the expression of WWP1 in various tissues and cell lines, including those known to express ENaC. As expected, WWP1 was expressed, like ENaC, in the bronchiolar epithelium. However it was also present in the proximal colon and the proximal part of the nephron (where ENaC is not expressed) and absent in the distal parts of the nephron (where ENaC is expressed abundantly). These results suggest that other channels or transport proteins, containing specific domains, such as PY motifs, could be the targets for regulation by WWP1.
Assuntos
Células Epiteliais/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Animais , Sequência de Bases , Células COS , Linhagem Celular , Chlorocebus aethiops , Complexos Endossomais de Distribuição Requeridos para Transporte , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Células LLC-PK1 , Masculino , Dados de Sequência Molecular , Ubiquitina-Proteína Ligases Nedd4 , Ratos , Ratos Sprague-Dawley , Suínos , Ubiquitina-Proteína Ligases/genética , Xenopus , Proteínas de XenopusRESUMO
Effects of aldosterone on its target cells have long been considered to be mediated exclusively through the genomic pathway; however, evidence has been provided for rapid effects of the hormone that may involve nongenomic mechanisms. Whether an interaction exists between these two signaling pathways is not yet established. In this study, the authors show that aldosterone triggers both early nongenomic and late genomic increase in sodium transport in the RCCD(2) rat cortical collecting duct cell line. In these cells, the early (up to 2.5 h) aldosterone-induced increase in short-circuit current (Isc) is not blocked by the mineralocorticoid receptor (MR) antagonist RU26752, it does not require mRNA or protein synthesis, and it involves the PKCalpha signaling pathway. In addition, this early response is reproduced by aldosterone-BSA, which acts at the cell surface and presumably does not enter the cells (aldo-BSA is unable to trigger the late response). The authors also show that MR is rapidly phosphorylated on serine and threonine residues by aldosterone or aldosterone-BSA. In contrast, the late (4 to 24 h) aldosterone-induced increase in ion transport occurs through activation of the MR and requires mRNA and protein synthesis. Interestingly, nongenomic and genomic aldosterone actions appear to be interdependent. Blocking the PKCalpha pathway results in the inhibition of the late genomic response to aldosterone, as demonstrated by the suppression of aldosterone-induced increase in MR transactivation activity, alpha1 Na(+)/K(+)/ATPase mRNA, and Isc. These data suggest cross-talk between the nongenomic and genomic responses to aldosterone in renal cells and suggest that the aldosterone-MR mediated increase in mRNA/protein synthesis and ion transport depends, at least in part, upon PKCalpha activation. E-mail: marcel.blot-chabaud@pharmacie.univ-mrs.fr
Assuntos
Aldosterona/farmacologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Proteína Quinase C/metabolismo , Receptores de Mineralocorticoides/metabolismo , Espironolactona/análogos & derivados , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Linhagem Celular , Túbulos Renais Coletores/efeitos dos fármacos , Antagonistas de Receptores de Mineralocorticoides , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa , Ratos , Receptor Cross-Talk/fisiologia , Soroalbumina Bovina/farmacologia , Transdução de Sinais/fisiologia , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Espironolactona/farmacologia , Transcrição Gênica/fisiologiaRESUMO
The regulation of plasma membrane Na(+)-K(+)-ATPases (NKA) expression by aldosterone and arginin vasopressin (AVP) in the cortical collecting duct (CCD) has been examined in a new rat CCD cell line, designated as RCCD(2). This cell line has maintained many characteristics of the CCD-in particular, the expression of the mineralocorticoid receptor. Mineralocorticoid receptor is expressed at the protein level and binds (3)H-aldosterone (approximately 15 to 20 fmol/mg protein). Short-circuit current (Isc) experiments showed approximately a twofold increase in Isc associated with a decrease in transepithelial resistance when cells were treated with aldosterone concentrations as low as 10(-9) M. This effect on Isc was significant 2 h after aldosterone addition and was still present after 24 h. It was accompanied by an increase in the amount of mRNA encoding for the alpha subunit of the epithelial sodium channel (sixfold) and the alpha1 subunit of NKA (fourfold) after 24 h of hormone treatment. In addition, mRNA expression of the serum- and glucocorticoid-induced kinase (Sgk) was increased by 10(-9) M aldosterone treatment as early as 45 min after hormone addition. As had already been documented in native CCD obtained by microdissection, incubation of RCCD(2) cells for 24 h with aldosterone resulted in the constitution of a latent pool of NKA that could be rapidly recruited by AVP (15 min). NKA biotinylation experiments and preparation of membrane fractions show that this latent pool of NKA is present in the intracellular compartment of the cells and is recruited by AVP in the basolateral membrane through a translocation process. This mechanism is accompanied by dephosphorylation of the alpha(1) catalytic subunit of NKA.
Assuntos
Aldosterona/fisiologia , Túbulos Renais Coletores/metabolismo , Proteínas Nucleares , ATPase Trocadora de Sódio-Potássio/metabolismo , Vasopressinas/fisiologia , Aldosterona/farmacologia , Amilorida/farmacologia , Animais , Arginina Vasopressina/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Biotina/metabolismo , Linhagem Celular , Condutividade Elétrica , Canais Epiteliais de Sódio , Proteínas Imediatamente Precoces , Córtex Renal , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/fisiologia , Ouabaína/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Sódio/metabolismo , Canais de Sódio/metabolismoRESUMO
The early phase of the stimulatory action of aldosterone on sodium reabsorption in tight epithelia involves hormone-regulated genes that remain to be identified. Using a subtractive hybridization technique on isolated renal cortical collecting ducts from rats injected with a physiological dose of aldosterone, we have identified an early response cDNA highly homologous to human and murine NDRG2 (N-Myc downstream regulated gene 2), which consists of four isoforms and belongs to a new family of differentiation-related genes. NDRG2 mRNA was expressed in classical aldosterone target epithelia, and in the kidney, it was specifically located in the collecting duct, the site of aldosterone-regulated sodium absorption. NDRG2 mRNA was increased within 45 min by aldosterone in the kidney and distal colon, whereas it was unaffected in the heart. In the RCCD2 collecting duct cell line, NDRG2 mRNA was enhanced as early as 15 min after aldosterone addition by transcription-dependent effects. NDRG2 was induced by aldosterone concentrations as low as 10(-9) M, and a maximal effect was observed at 10(-8) M. In contrast, the glucocorticoid dexamethasone was ineffective in NDRG2 expression, whereas the glucocorticoid-regulated gene sgk was induced. Taken together, these results indicate that NDRG2 regulation by aldosterone is an early mineralocorticoid-specific effect. Interestingly, NDRG2 is homologous to Drosophila MESK2, a component of the Ras pathway, suggesting that activation of the Ras cascade may play a significant role in mineralocorticoid signaling.
Assuntos
Aldosterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Mineralocorticoides/farmacologia , Proteínas/genética , Adrenalectomia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Colo/metabolismo , Primers do DNA , Hibridização In Situ , Córtex Renal/metabolismo , Cinética , Masculino , Dados de Sequência Molecular , Miocárdio/metabolismo , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Sódio/urina , Transcrição Gênica , Proteínas Supressoras de TumorRESUMO
Long-term effects of arginine vasopressin (AVP) in the kidney involve the transcription of unidentified genes. By subtractive hybridization experiments performed on the RCCD(1) cortical collecting duct cell line, we identified calcyclin as an early AVP-induced gene (1 h). Calcyclin is a calcium-binding protein involved in the transduction of intracellular signals. In the kidney, calcyclin was localized at the mRNA level in the glomerulus, all along the collecting duct, and in the epithelium lining the papilla. In RCCD(1) cells and in m-IMCD(3) inner medullary collecting duct cells, calcyclin was evidenced in the cytoplasm. Calcyclin mRNA levels were progressively increased by AVP treatment in RCCD(1) (1.7-fold at 4 h) and m-IMCD(3) (2-fold at 7.5 h) cells. In RCCD(1) cells, calcyclin protein levels were increased by 4 h of AVP treatment. In vivo, treatment of genetically vasopressin-deficient Brattleboro rats with AVP for 4 days induced an increase in both calcyclin and aquaporin-2 mRNA expression. Finally, introduction of anti-calcyclin antibodies into RCCD(1) cells by permeabilizing the plasma membrane prevented the long-term (but not short-term) increase in short-circuit current induced by AVP. Taken together, these results suggest that calcyclin is an early vasopressin-induced gene that participates in the late phase of the hormone response in transepithelial ion transport.