Stimulation of ENaC Activity by Rosiglitazone is PPARγ-Dependent and Correlates with SGK1 Expression Increase (original) (raw)
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Mechanisms of Regulation of Epithelial Sodium Channel by SGK1 in A6 Cells
The Journal of General Physiology, 2004
The serum and glucocorticoid induced kinase 1 (SGK1) participates in the regulation of sodium reabsorption in the distal segment of the renal tubule, where it may modify the function of the epithelial sodium channel (ENaC). The molecular mechanism underlying SGK1 regulation of ENaC in renal epithelial cells remains controversial. We have addressed this issue in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible system. Expression of a constitutively active mutant of SGK1 (SGK1TS425D) induced a sixfold increase in amiloride-sensitive short-circuit current (Isc). Using noise analysis we demonstrate that SGK1 effect onIscis due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability. Impedance analysis indicated that SGK1TS425Dincreased the absolute value of cell equivalent capacitance by an average of 13.7%. SGK1TS425Dalso produced a 1.6–1.9-fold increase in total and plasma...
Journal of Biological Chemistry, 1999
The serum-and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloridesensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of  and ␥ subunits of epithelial sodium channels (ENaCs) and causes an increase in the sgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by which sgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.
Pflügers Archiv - European Journal of Physiology
How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel’s α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to invitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC...
Regulation of sgk by aldosterone and its effects on the epithelial Na+ channel
American journal of physiology. Renal physiology
Aldosterone is the major corticosteroid regulating Na(+) absorption in tight epithelia and acts primarily by activating the epithelial Na(+) channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na(+) current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514-2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgk mRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na(+) channel activity. A point mutation in the beta-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.
Role of SGK in hormonal regulation of epithelial sodium channel in A6 cells
AJP: Cell Physiology, 2003
The purpose of this study was to examine the role of the serum-and glucocorticoidinduced kinase (SGK) in the activation of the epithelial sodium channel (ENaC) by aldosterone, arginine vasopressin (AVP), and insulin. We used a tetracycline-inducible system to control the expression of wild-type (SGK wt T ), constitutively active (S425D mutation; SGK S425D
In vivo phosphorylation of the epithelial sodium channel
Proceedings of the National Academy of Sciences, 1998
The activity of the epithelial sodium channel (ENaC) in the distal nephron is regulated by an antidiuretic hormone, aldosterone, and insulin, but the molecular mechanisms that mediate these hormonal effects are mostly unknown. We have investigated whether aldosterone, insulin, or activation of protein kinases has an effect on the phosphorylation of the channel. Experiments were performed in an epithelial cell line generated by stable cotransfection of the three subunits (␣, , and ␥) of ENaC. We found that  and ␥, but not the ␣ subunit, are phosphorylated in the basal state. Aldosterone, insulin, and protein kinases A and C increased phosphorylation of the  and ␥ subunits in their carboxyl termini, but none of these agents induced de novo phosphorylation of ␣ subunits. Serines and threonines but not tyrosines were found to be phosphorylated. The results suggest that aldosterone, insulin, and protein kinases A and C modulate the activity of ENaC by phosphorylation of the carboxyl termini of the  and ␥ subunits. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
British Journal of Pharmacology, 2012
BACKGROUND AND PURPOSE Glucocorticoids appear to control Na + absorption in pulmonary epithelial cells via a mechanism dependent upon serum and glucocorticoid-inducible kinase 1 (SGK1), a kinase that allows control over the surface abundance of epithelial Na + channel subunits (a-, band g-ENaC). However, not all data support this model and the present study re-evaluates this hypothesis in order to clarify the mechanism that allows glucocorticoids to control ENaC activity. EXPERIMENTAL APPROACH Electrophysiological studies explored the effects of agents that suppress SGK1 activity upon glucocorticoid-induced ENaC activity in H441 human airway epithelial cells, whilst analyses of extracted proteins explored the associated changes to the activities of endogenous protein kinase substrates and the overall/surface expression of ENaC subunits. KEY RESULTS Although dexamethasone-induced (24 h) ENaC activity was dependent upon SGK1, prolonged exposure to this glucocorticoid did not cause sustained activation of this kinase and neither did it induce a coordinated increase in the surface abundance of a-, band g-ENaC. Brief (3 h) exposure to dexamethasone, on the other hand, did not evoke Na + current but did activate SGK1 and cause SGK1-dependent increases in the surface abundance of a-, band g-ENaC. CONCLUSIONS AND IMPLICATIONS Although glucocorticoids activated SGK1 and increased the surface abundance of a-, band g-ENaC, these responses were transient and could not account for the sustained activation of ENaC. The maintenance of ENaC activity did, however, depend upon SGK1 and this protein kinase must therefore play an important but permissive role in glucocorticoid-induced ENaC activation. Abbreviations ENaC, epithelial sodium channel; IAmil, amiloride-sensitive component of the total membrane current; IAmil (-82 mV), amiloride-sensitive membrane current flowing at-82 mV; Im, total membrane current; Nedd-4/2, neural precursor cell expressed, developmentally down-regulated protein 4-2; NDRG1, protein encoded by n-myc downstream regulated gene 1; P70-S6K, 70 kDa ribosomal S6 kinase; PDK1, 3-phosphoinositide-dependent protein kinase 1; PIP2, phosphatidylinostiol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PKB, protein kinase B (also known as Akt); PRAS40, 40 kDa proline-rich substrate of Akt (PKB); SGK1, serum and glucocorticoid-inducible kinase 1; TORC1 and 2, target of rapamycin complex 1 and 2; VHold, holding potential; Vm, membrane potential
SPAK Sensitive Regulation of the Epithelial Na Channel ENaC
Kidney & blood pressure research, 2015
The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK participates in the regulation of NaCl and Na(+),K(+),2Cl(-) cotransport and thus renal salt excretion. The present study explored whether SPAK has similarly the potential to regulate the epithelial Na(+) channel (ENaC). ENaC was expressed in Xenopus oocytes with or without additional expression of wild type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK or catalytically inactive (D212A)SPAK, and ENaC activity estimated from amiloride (50 µM) sensitive current (Iamil) in dual electrode voltage clamp experiments. Moreover, Ussing chamber was employed to determine Iamil in colonic tissue from wild type mice (spak(wt/wt)) and from gene targeted mice carrying WNK insensitive SPAK (spak(tg/tg)). Iamil was observed in ENaC-expressing oocytes, but not in water-injected oocytes. In ENaC expressing oocytes Iamil was significantly increased following coexpression of wild-type SPAK and (T233E)SPAK, but ...