Scaffold protein connector enhancer of kinase suppressor of Ras isoform 3 (CNK3) coordinates assembly of a multiprotein epithelial sodium channel (ENaC)-regulatory complex.

Hormone regulation of ion transport in the kidney tubules is essential for fluid and electrolyte homeostasis in vertebrates. A large body of evidence has suggested that transporters and channels exist in multiprotein regulatory complexes; however, relatively little is known about the composition of these complexes or their assembly. The epithelial sodium channel (ENaC) in particular is tightly regulated by the salt-regulatory hormone aldosterone, which acts at least in part by increasing expression of the serine-threonine kinase SGK1. Here we show that aldosterone induces the formation of a 1.0-1.2-MDa plasma membrane complex, which includes ENaC, SGK1, and the ENaC inhibitor Nedd4-2, a key target of SGK1. We further show that this complex contains the PDZ domain-containing protein connector enhancer of kinase suppressor of Ras isoform 3 (CNK3). CNK3 physically interacts with ENaC, Nedd4-2, and SGK1; enhances the interactions among them; and stimulates ENaC function in a PDZ domain-dependent, aldosterone-induced manner. These results strongly suggest that CNK3 is a molecular scaffold, which coordinates the assembly of a multiprotein ENaC-regulatory complex and hence plays a central role in Na(+) homeostasis.

Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key role in the regulation of the epithelial sodium channel.

Aldosterone is the principal hormonal regulator of sodium homeostasis in vertebrates. It exerts its actions through the mineralocorticoid receptor (MR) that regulates the transcription of specific target genes. In recent years, a number of MR target genes have been identified that are involved in the regulation of the epithelial sodium channel (ENaC), a key modulator of renal sodium absorption. Here we report the identification of cnksr3 as a direct MR target gene that is up-regulated in response to physiological concentrations of aldosterone. The cnksr3 promoter exhibits two functional aldosterone-responsive regions, which were bound by the MR as assessed by chromatin immunoprecipitation (ChIP). In vivo, CNKSR3 was highly expressed in the renal cortical collecting duct (CCD), the prime target segment of aldosterone-regulated sodium retention in the kidney. CCD cell lines stably overexpressing or silencing CNKSR3 were electrophysiologically analyzed and show that CNKSR3 expression correlated with and is required for ENaC-mediated transepithelial sodium transport. In parallel, CNKSR3 expression led to decreased MEK phosphorylation. We conclude that CNKSR3, a homologue of scaffold proteins involved in MAPK pathway regulation, is a direct target of MR and is required for the maintenance of transepithelial sodium transport in the kidney.

A colonic mineralocorticoid receptor cell model expressing epithelial Na+ channels.

In the distal colon, the epithelial sodium channel (ENaC) is rate limiting for sodium absorption. Progress in the molecular characterization of ENaC expression and trafficking in response to the mineralocorticoid aldosterone has been hampered, since no epithelial colonic cell line existed expressing functional ENaC stimulated by nanomolar aldosterone via mineralocorticoid receptor (MR). Here, we present a human colonic epithelial cell line inducibly expressing the MR (HT-29/B6-Tet-On-MR) which exhibits aldosterone-dependent ENaC-mediated sodium transport in the presence of the short-chain fatty acid butyrate. Butyrate was necessary for high-level expression of MR which allowed for aldosterone-dependent upregulation of beta- and gamma-ENaC expression. As butyrate alone was not capable of promoting ENaC-mediated sodium transport, aldosterone-induced GILZ (glucocorticoid-induced leucine zipper protein) was identified as a candidate factor increasing apical ENaC levels.

Bendigoles A approximately C, new steroids from Gordonia australis Acta 2299.

Bendigoles A approximately C are the first secondary metabolites to be isolated from a member of the actinomycete genus Gordonia. They were detected in a culture filtrate extract of Gordonia australis Acta 2299 by HPLC-diode array analysis and characterized as new steroids by mass spectrometry and NMR experiments. Bendigole C show binding affinity to the human progesterone and A approximately C to androgen receptor but are inactive at mineralocorticoid and estrogen receptors. In in vitro transactivation studies bendigoles A and C showed moderate and weak androgenic activities.

Molecular pharmacology of the mineralocorticoid receptor: prospects for novel therapeutics.

The blockade of mineralocorticoid receptors (MR) has been shown to be an invaluable therapy in heart failure and hypertension. To date, only two steroidal antimineralocorticoids, spironolactone (and its active metabolite canrenone) and eplerenone, have been approved, whereas novel non-steroidal compounds are in preclinical and early development. The careful investigation of the efficacy and tolerance of spironolactone in essential hypertension initially supported the idea that a more selective second generation of MR antagonists is desired for chronic treatment of cardiovascular diseases. More than 40 years went by between the approval of the first MR antagonist spironolactone and the market introduction of its sole successor, eplerenone. The molecular pharmacology of MR antagonists may be addressed at different levels. Available preclinical and clinical data of the two approved steroidal antimineralocorticoids allow a good comparison of potency and selectivity of MR antagonists and their pharmacokinetic properties. The search for novel generations of MR antagonists with the ultimate goal of a more tissue selective mode of action may require novel compounds that are differentiated with respect to the binding mode to the MR. Other factors that may contribute to tissue selectivity as e.g. the physicochemical properties of a drug and how they influence the resulting pharmacology in the context of tissue selective co-factor expression are even less well understood. In the following we will review these aspects and demonstrate that the molecular pharmacology of current MR antagonists is on the one hand far from well understood and, on the other hand, still offers room for improvements.

Molecular basis for glucocorticoid induction of the Kruppel-like factor 9 gene in hippocampal neurons.

Stress has complex effects on hippocampal structure and function, which consequently affects learning and memory. These effects are mediated in part by circulating glucocorticoids (GC) acting via the intracellular GC receptor (GR) and mineralocorticoid receptor (MR). Here, we investigated GC regulation of Krüppel-like factor 9 (KLF9), a transcription factor implicated in neuronal development and plasticity. Injection of corticosterone (CORT) in postnatal d 6 and 30 mice increased Klf9 mRNA and heteronuclear RNA by 1 h in the hippocampal region. Treatment of the mouse hippocampal cell line HT-22 with CORT caused a time- and dose-dependent increase in Klf9 mRNA. The CORT induction of Klf9 was resistant to protein synthesis inhibition, suggesting that Klf9 is a direct CORT-response gene. In support of this hypothesis, we identified two GR/MR response elements (GRE/MRE) located -6.1 and -5.3 kb relative to the transcription start site, and we verified their functionality by enhancer-reporter, gel shift, and chromatin immunoprecipitation assays. The -5.3-kb GRE/MRE is largely conserved across tetrapods, but conserved orthologs of the -6.1-kb GRE/MRE were only detected in therian mammals. GC treatment caused recruitment of the GR, histone hyperacetylation, and nucleosome removal at Klf9 upstream regions. Our findings support a predominant role for GR, with a minor contribution of MR, in the direct regulation of Klf9 acting via two GRE/MRE located in the 5'-flanking region of the gene. KLF9 may play a key role in GC actions on hippocampal development and plasticity.

Glucocorticoid receptor is indispensable for physiological responses to aldosterone in epithelial Na+ channel induction via the mineralocorticoid receptor in a human colonic cell line.

The epithelial Na+ channel (ENaC) plays a crucial role in electrogenic Na(+) absorption in the distal colon. ENaC induction via the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) is differentially regulated by modulatory components. As most existing epithelial cell lines including colonic epithelial cell lines miss the co-expression of functional GR and MR, signaling on ENaC is only poorly characterized regarding the interplay of glucocorticoids and mineralocorticoids. In the present study, we show that GR expression and activity are indispensable for MR-dependent induction of ENaC-mediated Na(+) transport. Cooperativity of the two receptors has been studied in the highly differentiated, epithelial colonic cell line HT-29/B6-GR/MR which is equipped with the complete receptor repertoire of both GR and MR due to stable transfection. In contrast to HT-29/B6 cells solely expressing the MR, this cell line displays a physiological response to aldosterone regarding ENaC induction. To achieve this, a pre-incubation step with the GR agonist dexamethasone was required to allow for the subsequent stimulation of ENaC by aldosterone. As a result of cooperative effects between the activated GR and the MR, MR protein levels were elevated and MR-dependent transcription of ENaC subunits ß and γ was increased. As an additional mechanism involved, transcription of SGK-1 (serum- and glucocorticoid-induced kinase 1) and GILZ (glucocorticoid-induced leucin zipper)--both essential for the insertion of ENaC into the apical enterocyte membrane--were also augmented by the activated MR.