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The role of SGK1 in hormone-regulated sodium transport
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The role of SGK1 in hormoneregulated sodium transport David Pearce Ion transport in epithelia is regulated by a variety of hormonal and nonhormonal factors, including mineralocorticoids, insulin, shear stress and osmotic pressure. In mammals, the mineralocorticoid aldosterone is the principal regulator of sodium homeostasis and hence is central to the control of extracellular fluid volume and blood pressure. Aldosterone acts through a member of the nuclear receptor superfamily, the mineralocorticoid receptor (MR), to control the transcriptional activity of specific target genes. Recently, a serine/threonine kinase, SGK1 (serum and glucocorticoid-regulated kinase isoform 1) was identified as a candidate mediator of aldosterone action in the colon and distal nephron. The aldosterone-activated MR increases SGK1 gene transcription and SGK1, in turn, strongly stimulates the activity of the epithelial sodium channel (ENaC). Interestingly, other factors appear to regulate SGK1 gene expression and kinase activity. Insulin, for example, stimulates SGK1 activity (but not gene transcription) through its effects on phosphatidylinositol-3-kinase and osmotic shock appears to stimulate both SGK1 activity and gene transcription. Hence, SGK1 might integrate the effects of multiple hormonal and nonhormonal regulators of Na+ transport in tight epithelia and thereby play a key role in volume homeostasis. It is interesting to speculate that SGK1 might be implicated in medical conditions, such as the insulin resistance syndrome, hypertension and congestive heart failure.
David Pearce Division of Nephrology, Dept of Medicine and Dept of Cellular & Molecular Pharmacology, Box 0532, University of California, San Francisco, San Francisco, CA 94143, USA. e-mail: pearced@ medicine.ucsf.edu
Aldosterone is one of the key regulators of blood pressure (BP) and electrolyte homeostasis in vertebrates, owing, in large measure, to its effects on Na+ transport in tight epithelia. It binds with high affinity to the mineralocorticoid receptor (MR) and with lower but significant affinity to the glucocorticoid receptor (GR), both of which are members of the steroid receptor family of hormonedependent transcription factors (reviewed in Refs 1–3). Although MR is the principal mediator of aldosterone-stimulated ion transport in epithelia, it appears that GR contributes when aldosterone concentrations are high4,5. Hormone binding triggers a conformational change in all steroid and nuclear receptors that results in release of inactivating chaperones, translocation to the nucleus and/or changes in subnuclear architecture and regulation of specific target genes6,7. A substantial amount of recent effort in the steroid- and nuclear-receptor fields has focused on the protein–DNA and protein–protein interactions that lead to the formation of transcription preinitiation complexes and productive gene transcription. Hence, many of the general features of steroid and nuclear receptor regulation of gene expression in general, and MR and GR function in particular, have been well characterized (reviewed in Refs 1,2,8). However, until recently, the specific target genes and mechanisms involved in mineralocorticoid-regulated ion transport http://tem.trends.com
in epithelia remained obscure. Considerable progress has now been made in delineating the molecular pathways that link receptor-mediated changes in gene transcription with changes in ion transport. This review will focus on the role of the serum and glucocorticoid-regulated kinase (SGK) family, particularly isoform 1 (SGK1), in mineralocorticoidregulated Na+ transport, and will discuss the possible role of SGK1 as an integrator of multiple signals. The identification of novel isoforms of SGK (SGK2 and SGK3) and the possible role of the SGK family in the insulin resistance syndrome also will be addressed. Early effects of aldosterone in tight epithelia result from changes in gene transcription of a Na+ channel regulator
The physiological mechanisms underpinning aldosterone action, particularly in the kidney collecting duct (CD), have been extensively characterized in whole animals, isolated tubules and in cultured cells (reviewed in Ref. 9). In intact CD or cultured CD cells, the response to mineralocorticoids can be divided into two main phases – early and late – that differ significantly, although both require changes in gene transcription (reviewed in Ref. 3). The principal early action of aldosterone in CD cells (starting after a latent period of 30–45 min) is to increase apical membrane permeability by increasing Na+ transport through the epithelial Na+ channel (ENaC)9,10. The ENaC is probably a heterotetramer composed of three different subunits (α, β and γ) in the ratio 2:1:1 (Ref. 11). It is expressed in a variety of Na+-transporting epithelia, including lung, colon and parotid gland, in addition to CD, where it constitutes the rate-limiting step in transepithelial Na+ transport. The biophysical and physiological properties of the ENaC have been discussed extensively in numerous reviews (e.g. Ref. 9). One aspect of its regulation that warrants particular comment in the present context is the regulatory action of the ubiquitin ligase, Nedd4 (neural precursor cell expressed, developmentally downregulated 4). Nedd4 was originally identified in a subtracted library representing genes downregulated during development of the mouse brain12. Although its function in neural development remains uncertain, it was subsequently independently cloned as an interacting protein for the C-terminal tail of the ENaCβ subunit13. Analysis of a series of truncations and point mutations revealed
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Fig. 1. Northern blot analysis revealing rapid and transient induction of SGK1 mRNA synthesis in A6 cells treated with dexamethasone. Blots were hybridized with a probe for Xenopus SGK1 and type 8 actin29. Dexamethasone is the most potent stimulus of SGK1 expression in A6 cells; aldosterone gives a similar but less robust effect. Reproduced, with permission, from Ref. 29.
that a tryptophan-rich region of Nedd4 (‘WW domain’) stably interacts with a proline- and tyrosinerich amino stretch in Nedd4 termed the ‘PY’ motif. Following Nedd4-dependent ubiquitination, channel proteins are removed from the plasma membrane and either recycled or undergo proteasome-mediated degradation. Interestingly, Liddle’s syndrome, a form of pseudoaldosteronism, results from ENaC mutations that disrupt the PY motif and interaction with Nedd4 (Refs 13–15). In most cases, the ENaC-mediated early effect of aldosterone accounts for more than 60% of the total increase in Na+ current3 and, importantly, occurs without changes in ENaC gene transcription or mRNA levels16–24. Since aldosterone action clearly requires changes in gene transcription, these observations suggested the hypothesis that a key mineralocorticoid-regulated gene (or genes) encoded a regulatory protein that increases the plasma membrane localization and/or activity of already synthesized ENaC subunits. Nevertheless, regulation of α-subunit biosynthesis might be important for the late effects of aldosterone20,24. These considerations prompted several groups to search for ENaC regulators as mediators of the early aldosterone effect. Many candidates, however, had pleiotrophic effects or were only modestly or slowly stimulated9,25–28. The early effects of aldosterone on Na+ transport are potent, rapid and largely limited to changes in ENaC-mediated Na+ transport; other effects, such as changes in the proliferative state of CD cells or increases in membrane surface area, occur later29. Thus, the mRNA and protein levels of a mediator of the early response should increase markedly and rapidly and the mediator should, in turn, strongly stimulate ENaC activity. Recently, SGK1 has been shown to be just such an aldosteronestimulated regulator of ENaC activity30–33. SGK1 mRNA is rapidly increased by mineralocorticoids and, when expressed in Xenopus oocytes, SGK1 strongly and selectively stimulates ENaC-mediated Na+ transport in addition to the localization of ENaC in the membrane. Interestingly, SGK1 activity (but not abundance) is regulated by insulin, suggesting http://tem.trends.com
SGK1 was originally identified as a glucocorticoidregulated mRNA in mammary epithelial cells and termed SGK to reflect its regulation at the transcriptional level by serum and glucocorticoids34. It was subsequently cloned by several labs and its expression level was found to be regulated by a variety of hormonal and non-hormonal stimuli, including serum, follicle stimulating hormone (FSH)35, osmotic shock36 and mineralocorticoids30–32. It has the characteristic motifs of a serine/threonine kinase and kinase activity has been demonstrated in vitro37–39. SGK1 is found in most vertebrate tissues, although at variable levels34, and its expression pattern within tissues shows considerable heterogeneity with respect to both basal and stimulated levels of expression31. Thus, in kidney CD, basal levels of SGK1 are low and are strongly induced by mineralocorticoids, whereas in proximal tubule, expression is undetectable (by in situ hybridization) in the absence or presence of mineralocorticoids or glucocorticoids. In glomerular mesangial cells, moderate basal levels of SGK1 are detected that appear to be uninfluenced by corticosteroids. Interestingly, TGFβ (transforming growth factor β) could be a potent stimulator of SGK1 expression in this cell type40. The regulation of SGK1 expression in other tissues is similarly cell-type specific36,41. SGK1 mRNA and protein levels are regulated by mineralocorticoids in CD and colon
In an attempt to identify candidate mineralocorticoid response genes involved in the control of Na+ transport, our lab used suppression subtractive hybridization to generate a cDNA library enriched in dexamethasone-induced gene products in A6 cells42. From the resulting library, one clone showed very high homology to rat and human SGK1 and was selected for further characterization. Northern blot analysis confirmed rapid and transient dexamethasone induction of SGK1 mRNA in A6 cells (Fig. 1), and not surprisingly, induction of SGK1 mRNA was not blocked by cycloheximide30, suggesting that it has a direct transcriptional effect. Although dexamethasone was used to generate the library because of the substantially higher levels of GR than MR in these cells43,44, high concentrations of aldosterone, sufficient to activate GR, similarly stimulated SGK1 gene expression (unpublished). It is important to note that ‘glucocorticoids’ can have ‘mineralocorticoid’ effects in classical mineralocorticoid target cells45: their failure to act as mineralocorticoids does not depend on receptor specificity, but rather on the action of an enzyme, 11β-hydroxysteroid dehydrogenase46. Because this
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Fig. 2. In situ hybridization of SGK1 in rat kidney. Adrenalectomized rats were treated for 4 h with (a) vehicle and then hybridized with sense SGK1 mRNA, (b) vehicle and then hybridized with anti-sense SGK1 mRNA and (c) vehicle + aldosterone and hybridized with anti-sense SGK1 mRNA (Ref. 29). Note the appearance of the cortex in (c), suggesting ‘medullary rays’. Inspection of emulsion-dipped sections confirmed that these are indeed cortical collecting ducts. Abbreviation: SGK1, serum and glucocorticoid-regulated kinase isoform 1. Reproduced, with permission, from Ref. 29.
issue continues to cause confusion, many investigators use superphysiological doses of aldosterone to stimulate Na+ transport and infer that the effect is more reflective of true mineralocorticoid physiology than if they used dexamethasone or another ‘glucocorticoid’. For reasons that remain uncertain, most stable cell lines lose MR expression5,43,44,47. In these cells, the effects of aldosterone are mediated primarily, if not solely, by GR (hence the high concentrations of aldosterone required to stimulate Na+ currents)4. In CD cells and possibly other Na+-transporting epithelia that express both MR and GR, both receptors mediate similar, if not identical effects on gene transcription and on ion transport44,45. Hence, the term ‘mineralocorticoid’ (defined functionally) is appropriate for dexamethasone in this context. In contexts where MR and GR mediate strikingly different effects, the distinction is mechanistically extremely important49,50. It seems likely that the semantic confusion that still surrounds this topic will become clearer as the mechanisms underlying mineralocorticoid action are fully elucidated. Regardless of terminology, conclusions drawn from artificial systems must be verified in systems that are (a)
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Fig. 3. SGK1 stimulates ENaC but not ROMK2 activity. (a) In vitro transcribed RNA for each of the three ENaC subunits was injected into Xenopus laevis oocytes with or without SGK1. Shown is amiloride-sensitive current (Iami). (b) In vitro transcribed ROMK2 mRNA was injected into Xenopus oocytes with or without SGK1, as in (a). ROMK2 is the principal potassium channel found in collecting duct apical membrane. Abbreviations: ENaC, epithelial Na+ channel; ROMK2, principal collecting duct K+ channel; SGK1, serum and glucocorticoid-regulated kinase isoform 1. Reproduced, with permission, from Ref. 29.
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of clear physiological relevance. Importantly, aldosterone has been shown to stimulate SGK1 expression in both the CD of intact animals30,50, and in primary cultures of CD cells31, which express both MR and GR at high levels45. Our lab used in situ hybridization to show that SGK1 is induced by aldosterone in the CD of rat kidney (Fig. 2). The response of SGK1 to the physiological mineralocorticoid aldosterone was rapid, beginning within 0.5 h of a single injection of aldosterone; its levels also decreased rapidly, returning to near baseline by 24 h (Refs 30,50; unpublished), consistent with the short half-life of both aldosterone and SGK1 (Ref. 51). Both the time course and dose response of SGK1 to aldosterone correlate well with aldosteroneinduced changes in Na+ and K+ transport50. SGK1 stimulates ENaC-mediated Na+ transport and plasma membrane localization
ENaC constitutes the rate-limiting step for Na+ transport in aldosterone-responsive epithelia and is the principal early target of aldosterone action9,18. Consistent with an important role in mediating the early effects of aldosterone, SGK1 strongly stimulates ENaC-mediated Na+ currents in a Xenopus oocyte coexpression assay30–33 (Fig. 3). Importantly, as noted above, a kinase-dead mutant of SGK1 does not stimulate ENaC and indeed, has dominant negative activity52. Furthermore, SGK1 does not stimulate principal collecting duct K+ channel (ROMK2)mediated K+ transport, an observation that is of interest, not only because it suggests specificity to the SGK1 effect, but also because ROMK2 is the major pathway for K+ transport in CD. This observation is consistent with the view that changes in K+ transport depend on alterations in the electrochemical gradient established by Na+ movement through the ENaC, although additional regulators could be implicated. Interestingly, recent evidence supports the possibility that SGK1 does stimulate a K+ channel unrelated to ROMK2, Kv1.3*. The significance of this observation, however, remains to be determined. Initial insight into the mechanism of SGK1 stimulation of ENaC is provided by the observation that the kinase increases ENaC plasma membrane localization to an extent comparable with its stimulation of Na+ current, when expressed in Xenopus oocytes52,53 (Fig. 4). This observation is particularly relevant in the light of mounting evidence that aldosterone stimulates ENaC-mediated Na+ transport by increasing its apical membrane localization24,52. Importantly, SGK1 kinase activity is required, both for stimulation of Na+ current, and for increased ENaC plasma membrane localization50 (Fig. 4). Indeed, kinase-dead forms of SGK1 have dominant negative activity, probably because they *Beck, S. et al. (2001) The aldosterone-regulated serine/threonine kinase SGK1 stimulates the Kv1.3 potassium channel. Joint Meeting of the Scandinavian Physiological Society and of the German Physiological Society
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displace wild-type SGK1 from cellular targets. In contrast to its effect on plasma membrane localization, SGK1 appears to have little or no effect on open probability of the channels53; however, this issue remains unsettled and it is possible that under physiological conditions, SGK1 could impact on open probability, as has been suggested for aldosterone16.
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Fig. 4. Wild-type (wt) SGK1 stimulates ENaC-mediated current (a) and ENaC surface localization (b,c) in Xenopus laevis oocytes. A kinase-dead mutant (mut SGK) has the opposite effect50. (a) Either the wild type or K130A mutant (unable to bind ATP) of SGK1 was coexpressed with ENaC subunits in Xenopus oocytes and amiloride-sensitive current (Iami) was measured by two-electrode voltage clamp. (b) Surface expression of FLAG-epitope tagged ENaC subunits was detected by binding of radioiodinated anti-FLAG antibody. (c) FLAG–ENaC surface expression detected by immunostaining in (i) control, (ii) wt SGK and (iii) mut SGK oocytes. Abbreviations: ENaC, epithelial Na+ channel; SGK, serum and glucocorticoid-regulated kinase. Reproduced, with permission, from Ref. 50.
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Although aldosterone is the main physiological regulator of CD Na+ transport, insulin also stimulates reabsorption54–56, a process that might be of considerable relevance to the pathogenesis of hypertension associated with the insulin resistance syndrome (‘syndrome X’)57. Interestingly, the closest relative of SGK1 (other than SGK2 and 3) is protein kinase B (PKB)/Akt, a member of the serine/threonine kinase family, which plays a central role in insulin signaling and has been studied extensively58. PKB/Akt is activated by phosphatidylinositol-3-kinase (PI3K) through phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent kinase 1 (PDK1)59. SGKs of all species, including yeast, have PDK consensus sites like those of PKB/Akt; recent evidence indicates that they are phosphorylation targets of PDK1 (and possibly PDK2)38,60, and that their activities are PI3Kdependent37. Importantly, blockade of PI3K not only abolishes insulin-stimulated Na+ transport61 and the early component of mineralocorticoid-stimulated Na+ transport, but also eliminates phosphorylation of SGK162,63 (Fig. 5a,b). Furthermore, insulin, which has no effect on SGK1 gene expression, augments SGK1 activity by increasing its phosphorylation37,60,63 (Fig. 5c). Under these conditions, mineralocorticoids and insulin have been found to stimulate Na+ transport in CD cells synergistically63–65 (Fig. 5c). These observations suggest that SGK1 is an integrator of insulin and mineralocorticoids in the control of Na+ transport, as shown schematically in Fig. 6. However, aldosterone–insulin synergy has not always been found66.
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Fig. 5. (a) Amiloride-inhibitable equivalent current, which represents ENaC-mediated Na+ transport, showing blocking of the early mineralocorticoid effect on Na+ transport by PI3K inhibition in A6 cells. Cells were treated with the specific PI3K inhibitor, LY294002 (circles), or nothing (squares), before addition of dexamethasone after 0.5 h. PI3K inhibitor + dexamethasone (triangles) and dexamethasone only (diamonds). Where not shown, error bars were smaller than data symbols. (b) Immunoblots probed for SGK1 at different times, demonstrating block of SGK1 phosphorylation by PI3K inhibition in A6 cells. Cells were treated (i) without and (ii) with PI3K inhibitor and the upper and lower bands are the phosphorylated and unphosphorylated forms of SGK1, respectively. (c) Amiloride-inhibitable equivalent current, showing synergy between insulin, which increases phosphorylation of SGK1, and dexamethasone in stimulating ENaC-mediated Na+ transport in A6 cells. Cells were treated with dexamethasone (or vehicle) alone for 2 h, followed by addition of insulin (or vehicle) for an additional 0.5 h and then harvested and immunoblotted for SGK1. Changes in equivalent current relative to that of epithelia untreated with dexamethasone or insulin were (in µA cm−2): 4.1 (dexamethasone alone); 5.8 (insulin alone); 15.3 (dexamethasone + insulin). The change in equivalent current induced by insulin and dexamethasone together was significantly greater than the sum of those induced by the two separately (15.3 versus 9.9 µA; P < 0.001, unpaired Student’s ttest). Ratio of phosphorylated to unphosphorylated SGK1 was significantly higher in the presence of insulin (P < 0.01, unpaired Student’s t-test), but was unaffected by dexamethasone. Abbreviations: Dex, dexamethasone; ENaC, epithelial Na+ channel; Ins, insulin; PI3K, phosphatidylinositol-3-kinase; SGK1, serum and glucocorticoid-regulated kinase isoform 1. Reproduced, with permission, from Ref. 29.
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SGK1 is a candidate integrator of insulin and aldosterone effects on Na+ transport
Other regulators of SGK1 activity and/or expression
A variety of other factors impact on SGK1 activity and/or expression. Osmotic shock, for example, potently stimulates SGK1 gene transcription36,67,68, whereas insulin stimulates its activity40 and FSH stimulates both35,69. Osmotic shock is a particularly intriguing stimulus of SGK1 expression in the present context because it is the most ancient effector of changes in cell volume, and an important determinant of the homeostatic response of freshwater and euryhaline vertebrates to changes in the ionic composition of their environment. In early (Precambrian) marine organisms, SGK1 might have been regulated by osmotic strength (possibly through changes in cell volume), with other regulators introduced later, most notably for our purposes, the renin–angiotensin–aldosterone system, which is first
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Fig. 6. A speculative scheme of hormone-regulated Na+ transport, emphasizing the role of SGK1. According to this scheme, SGK1 abundance is regulated by mineralocorticoids, whereas its activity is regulated by PI3K (through PIP3 and PDK1). SGK2 and SGK3 activities are thought to be similarly regulated through PI3K; however, their levels do not appear to be regulated by mineralocorticoids or glucocorticoids. Before its interaction with SGK1, ENaC is shown in a hypothetical vesicle, analogous to insulin-regulated vesicles. The dotted line between PIP3 and PDK1 indicates the stable, non-covalent interaction between PIP3 and the PH domain of PDK1. Abbreviations: ECF, extracellular fluid; ENaC, epithelial Na+ channel; Ins, insulin; IRS1, insulin receptor substrate 1; IRV, insulin-regulated vesicle; MC, mineralocorticoid; MR, mineralocorticoid receptor; PDK1, PIP3-dependent kinase 1; PH, pleckstrin homology domain; PI3K, phosphatidylinositol-3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; ROMK2, principal collecting duct K+ channel; SGK1, serum and glucocorticoidregulated kinase isoform 1. Reproduced, with permission, from Ref. 29.
seen in amphibia or possibly (in a rudimentary form) in bony fish70. A teleological reason for the regulation of SGK1 by other hormones such as insulin, TGFβ and FSH is less clear. This is, in part, because until recently, the only clearly established physiological role for SGK1 was in the control of epithelial ion transport71,72. However, recent evidence has begun to provide direct support for the idea that SGK1, in some settings, might be implicated in the control of proliferation and apoptosis, as suggested previously34. Thus, SGK1 was recently shown to phosphorylate and inactivate the Forkhead family member, FKHRL1, a proapoptotic transcription factor previously known to be a regulatory target of Akt/PKB (Refs 39,73). In addition to this powerful, albeit indirect, evidence, one recent report provided direct evidence that SGK1 can be antiapoptotic74. Furthermore, another recent report directly demonstrated that phosphorylation of SGK1 at Ser78 by the mitogen activated kinase BMK1 is http://tem.trends.com
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Other SGK isoforms
Recently, two additional isoforms of SGK were identified60. Hence, the originally identified, glucocorticoid–mineralocorticoid-regulated form of SGK is now referred to as SGK1. Neither of these novel isoforms is glucocorticoid-regulated, and preliminary data suggest that they are not mineralocorticoid regulated either (D. Pearce, unpublished). The three isoforms of SGK share significant nucleotide (~60%) and amino acid (~80%) identity in their kinase domains and potential for misidentification and functional cross talk exists. SGK3, like SGK1, is expressed in all tissues examined by northern blot, whereas SGK2 is expressed only in kidney, liver, pancreas and brain. The distribution of SGK2 and SGK3 within tissues has not been examined and their physiological roles are unknown. However, it has been established that both stimulate ENaC-mediated Na+ transport†. A mechanistic view of SGK-stimulated Na+ transport
SGK1 stimulation of ENaC-mediated Na+ transport clearly requires its kinase activity52 (Fig. 4). However, in spite of attempts by several investigators, phosphorylation of ENaC subunits by SGK1 has not been demonstrated51 (D. Pearce, unpublished; F. Lang, pers. commun.). Moreover, SGK1 and probably SGK2 and SGK3 increase ENaC localization to the plasma membrane, although an effect on channel open probability has not been ruled out. Hence, it seems likely that the target proteins of the SGKs are involved in ENaC translocation to the plasma membrane, its removal, or both. Although there is some evidence suggesting that ENaC removal is not reduced by SGK1 (Ref. 53), additional evidence is needed to settle this issue. In principle, the most efficient way to achieve the rapid and potent effects that aldosterone and SGK1 have on ENaC is to alter both insertion and removal of channels. With regard to regulation of channel insertion, striking parallels between glucose transporter (GLUT4)-mediated glucose transport and ENaC-mediated Na+ transport have become apparent, perhaps reflecting common themes that underlie the hormonal control of protein trafficking events via intracellular signaling kinases76,77. Although the principal PKB/Akt targets involved in the exocytotic process have not been identified, recently, Synip, an insulin-regulated syntaxin 4-interacting protein, was identified as a potential regulator of GLUT4 vesicle fusion78. †Friederich, B. et al. (2000) Serine/Threonine kinases SGK2 and SGK3 both upregulate renal epithelial Na+-channel α, β, γ ENaC. American Society of Nephrology Annual Meeting Abstr. 149
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Acknowledgements Work performed in the author’s laboratory was supported by NIH grants DK51151 and DK56695 as well as a Grant-in-Aid from the American Heart Association Western States Affiliate. Gary Firestone is gratefully acknowledged for suggestions on the manuscript. Florian Lang and Olivier Staub are gratefully acknowledged for communicating unpublished data.
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With regard to mineralocorticoid regulation of channel removal, it is interesting to return to Liddle’s syndrome, which probably results from disruption of ENaC interaction with Nedd4 (Refs 13,14,79). In light of the opposing actions of SGK and Nedd4 on ENaC-mediated Na+ transport and the clinical similarities between Liddle’s syndrome and primary aldosteronism, it is plausible that SGK1 might influence Nedd4 activity, or vice versa. Importantly, since aldosterone does not regulate Nedd4 gene transcription80, any role that Nedd4 plays in aldosterone action must be indirect or as a target of an aldosterone-regulated gene product, such as SGK. According to the speculative scheme shown in Fig. 6, both phosphorylated and unphosphorylated forms of SGK1 are recruited to ENaC-containing vesicles by the channel itself, as suggested by recent evidence that SGK1 physically interacts with ENaC subunits, in vitro63. The activated form of SGK1 (phosphorylated at Ser256 and Ser422; human numbering) then phosphorylates proteins (possibly Synip or a Synip-like protein) that regulate channel
References 1 Yamamoto, K.R. et al. (1992) Combinatorial regulation at a mammalian composite response element. In Transcriptional Regulation (Vol. 22) (McKnight, S.L. and Yamamoto, K.R., eds), pp. 1169–1192, Cold Spring Harbor Press 2 Lin, R.J. et al. (1998) The transcriptional basis of steroid physiology. Cold Spring Harbor Symp. Quant. Biol. 63, 577–585 3 Verrey, F. (1995) Transcriptional control of sodium transport in tight epithelial by adrenal steroids. J. Membr. Biol. 144, 93–110 4 Rousseau, G. et al. (1972) Glucocorticoid and mineralocorticoid receptors for aldosterone. J. Steroid. Biochem. 3, 219–227 5 Bens, M. et al. (1999) Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line. J. Am. Soc. Nephrol. 10, 923–934 6 Fejes-Toth, G. et al. (1998) Subcellular localization of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists. Proc. Natl. Acad. Sci. U. S. A. 95, 2973–2978 7 DeFranco, D.B. (1999) Regulation of steroid receptor subcellular trafficking. Cell Biochem. Biophys. 30, 1–24 8 Xu, L. et al. (1999) Coactivator and corepressor complexes in nuclear receptor function. Curr. Opin. Genet. Dev. 9, 140–147 9 Garty, H. and Palmer, L.G. (1997) Epithelial sodium channels: function, structure, and regulation. Physiol. Rev. 77, 359–396 10 Verrey, F. (1999) Early aldosterone action: toward filling the gap between transcription and transport. Am. J. Physiol. 277, F319–F327 11 Firsov, D. et al. (1998) The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO J. 17, 344–352 12 Kumar, S. et al. (1992) Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185, 1155–1161 13 Staub, O. et al. (1996) WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ http://tem.trends.com
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insertion (via vesicle fusion with the plasma membrane) and possibly removal (for example Nedd4). The resulting increase in ENaC present at the apical membrane largely accounts for the increase in Na+ transport, although coincident changes in channel-open probability might also occur. This scheme accounts for the dominant negative activity of kinase-dead forms of SGK1, which interact with ENaC nonproductively, displacing wild-type kinase52. The dual regulation of SGK1 – of its abundance through a transcriptional mechanism and of its activity through a PI3K-dependent pathway – provides a mechanism for integrating multiple different inputs that impact on Na+ influx into epithelial cells, particularly those of aldosterone and insulin63. It will be of considerable interest for future investigation to determine if SGK1 plays a role in the salt-sensitive hypertension associated with the insulin-resistance syndrome57. Furthermore, the roles of SGK2 and SGK3 in normal signaling and disease pathogenesis remain to be characterized.
channel deleted in Liddle’s syndrome. EMBO J. 15, 2371–2380 Shimkets, R.A. et al. (1994) Liddle’s syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel. Cell 79, 407–414 Schild, L. et al. (1996) Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. EMBO J. 15, 2381–2387 Kemendy, A.E. et al. (1992) Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia. Am. J. Physiol. 263, C825–C837 Kleyman, T.R. et al. (1992) Aldosterone does not alter apical cell-surface expression of epithelial Na+ channels in the amphibian cell line A6. J. Biol. Chem. 267, 9622–9628 Eaton, D.C. et al. (1995) Renal sodium channels: regulation and single channel properties. Kidney Int. 48, 941–949 Ling, B.N. et al. (1997) Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line. J. Biol. Chem. 272, 594–600 May, A. et al. (1997) Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells. J. Am. Soc. Nephrol. 8, 1813–1822 Asher, C. et al. (1996) Aldosterone-induced increase in the abundance of Na+ channel subunits. Am. J. Physiol. 271, C605–C611 Denault, D. L. et al. (1996) Aldosterone regulation of sodium channel gamma-subunit mRNA in cortical collecting duct cells. Am. J. Physiol. 271, C423–C428 Escoubet, B. et al. (1997) Noncoordinate regulation of epithelial Na channel and Na pump subunit mRNAs in kidney and colon by aldosterone. Am. J. Physiol. 272, C1482–C1491 Masilamani, S. et al. (1999) Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney. J. Clin. Invest. 104, R19–R23 Sariban-Sohraby, S. et al. (1984) Methylation increases sodium transport into A6 apical
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37 Park, J. et al. (1999) Serum and glucocorticoidinducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J. 18, 3024–3033 38 Kobayashi, T. and Cohen, P. (1999) Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem. J. 339, 319–328 39 Brunet, A. et al. (2001) Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol. Cell. Biol. 21, 952–965 40 Waldegger, S. et al. (1999) h-sgk serine-threonine protein kinase gene as transcriptional target of transforming growth factor beta in human intestine. Gastroenterology 116, 1081–1088 41 Imaizumi, K. et al. (1994) Differential expression of sgk mRNA, a member of the Ser/Thr protein kinase gene family, in rat brain after CNS injury. Brain Res. Mol. Brain Res. 26, 189–196 42 Diatchenko, L. et al. (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissuespecific cDNA probes and libraries. Proc. Natl. Acad. Sci. U. S. A. 93, 6025–6030 43 Schmidt, T.J. et al. (1993) Steroid hormone stimulation of Na+ transport in A6 cells is mediated via glucocorticoid receptors. Am. J. Physiol. 264, C875–C884 44 Chen, S.Y. et al. (1998) Aldosterone responsiveness of A6 cells is restored by cloned rat mineralocorticoid receptor. Am. J. Physiol. 274, C39–C46 45 Naray-Fejes-Toth, A. and Fejes, G. (1990) Glucocorticoid receptors mediate mineralocorticoid-like effects in cultured collecting duct cells. Am. J. Physiol. 259, F672–F678 46 Funder, J.W. et al. (1988) Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242, 583–585 47 Beaman-Hall, C.M. et al. (1996) Expression of glucocorticoid and mineralocorticoid receptors in an immortalized hippocampal neuronal cell line. Brain Res. 726, 141–152 48 Joels, M. and de Kloet, E.R. (1990) Mineralocorticoid receptor-mediated changes in membrane properties of rat CA1 pyramidal neurons in vitro. Proc. Natl. Acad. Sci. U. S. A. 87, 4495–4498 49 Pearce, D. and Yamamoto, K.R. (1993) Mineralocorticoid and glucocorticoid receptor activities distinguished by nonreceptor factors at a composite response element. Science 259, 1161–1165
50 Bhargava, A. et al. (2001) The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action. Endocrinology 142, 1587–1594 51 Webster, M.K. et al. (1993) Immediate-early transcriptional regulation and rapid mRNA turnover of a putative serine/threonine protein kinase. J. Biol. Chem. 268, 11482–11485 52 Loffing, J. et al. (2001) Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK. Am. J. Physiol. 280, F675–F682 53 Alvarez de la Rosa, D. et al. (1999) The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes. J. Biol. Chem. 274, 37834–37839 54 DeFronzo, R.A. et al. (1976) The effects of glucose and insulin on renal electrolyte transport. J. Clin. Invest. 58, 83–90 55 Fidelman, M.L. et al. (1982) Insulin stimulation of Na+ transport and glucose metabolism in cultured kidney cells. Am. J. Physiol. 242, C121–C123 56 Kageyama, S. et al. (1994) Effect of insulin on sodium reabsorption in hypertensive patients. Am. J. Hypertens. 7, 409–415 57 Reaven, G.M. (1997) The kidney: an unwilling accomplice in syndrome X. Am. J. Kidney Dis. 30, 928–931 58 Downward, J. (1998) Mechanisms and consequences of activation of protein kinase B/Akt. Curr. Opin. Cell Biol. 10, 262–267 59 Shepherd, P.R. et al. (1998) Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. Biochem. J. 333, 471–490 60 Kobayashi, T. et al. (1999) Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem. J. 344, 189–197 61 Record, R.D. et al. (1998) Phosphatidylinositol 3-kinase activation is required for insulinstimulated sodium transport in A6 cells. Am. J. Physiol. 274, E611–E617 62 Blazer-Yost, B.L. et al. (1999) Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption. Am. J. Physiol. 277, C531–C536 63 Wang, J. et al. (2001) SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport. Am. J. Physiol. 280, F303–F313 64 Fidelman, M.L. and Watlington, C.O. (1984) Insulin and aldosterone interaction on Na+ and K+ transport in cultured kidney cells (A6). Endocrinology 115, 1171–1178 65 Johnson, J.P. et al. (1986) Hormonal regulation of Na+–K+-ATPase in cultured epithelial cells. Am. J. Physiol. 251, C186–C190
66 Record, R.D. et al. (1996) Aldosterone and insulin stimulate amiloride-sensitive sodium transport in A6 cells by additive mechanisms. Am. J. Physiol. 271, C1079–C1084 67 Bell, L.M. et al. (2000) Hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) by a p38 MAPK-dependent pathway. J. Biol. Chem. 275, 25262–25272 68 Waldegger, S. et al. (1998) Cloning of sgk serine–threonine protein kinase from shark rectal gland – a gene induced by hypertonicity and secretagogues. Pflügers Arch. 436, 575–580 69 Gonzalez-Robayna, I.J. et al. (2000) Folliclestimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoidinduced kinase (Sgk): evidence for A kinaseindependent signaling by FSH in granulosa cells. Mol. Endocrinol. 14, 1283–1300 70 Wilson, J.X. (1984) The renin–angiotensin system in nonmammalian vertebrates. Endocr. Rev. 5, 45–61 71 Pearce, D. et al. (2000) Role of SGK in mineralocorticoid-regulated sodium transport. Kidney Int. 57, 1283–1289 72 Cole, T.J. and Pearce, D. Mineralocorticoid target genes. Curr. Opin. Endocrinol. Diabetes (in press) 73 Brunet, A. et al. (1999) Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 96, 857–868 74 Mikosz, C.A. et al. (2001) Glucocorticoid receptor-mediated protection from apoptosis is associated with induction of the serine/threonine survival kinase gene, sgk-1. J. Biol. Chem. 276, 16649–16654 75 Hayashi, M. et al. (2001) BMK1 mediates growth factor-induced cell proliferation through direct cellular activation of serum and glucocorticoid-inducible kinase. J. Biol. Chem. 276, 8631–8634 76 James, D.E. et al. (1993) Targeting of mammalian glucose transporters. J. Cell. Sci. 104, 607–612 77 Pessin, J.E. et al. (1999) Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. Location! Location! Location! J. Biol. Chem. 274, 2593–2596 78 Min, J. et al. (1999) Synip: a novel insulinregulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes. Mol. Cell 3, 751–760 79 Staub, O. et al. (2000) Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination. Kidney Int. 57, 809–815 80 Staub, O. et al. (1997) Immunolocalization of the ubiquitin-protein ligase Nedd4 in tissues expressing the epithelial Na+ channel (ENaC). Am. J. Physiol. 272, C1871–C1880
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The role of SGK1 in hormoneregulated sodium transport David Pearce Ion transport in epithelia is regulated by a variety of hormonal and nonhormonal factors, including mineralocorticoids, insulin, shear stress and osmotic pressure. In mammals, the mineralocorticoid aldosterone is the principal regulator of sodium homeostasis and hence is central to the control of extracellular fluid volume and blood pressure. Aldosterone acts through a member of the nuclear receptor superfamily, the mineralocorticoid receptor (MR), to control the transcriptional activity of specific target genes. Recently, a serine/threonine kinase, SGK1 (serum and glucocorticoid-regulated kinase isoform 1) was identified as a candidate mediator of aldosterone action in the colon and distal nephron. The aldosterone-activated MR increases SGK1 gene transcription and SGK1, in turn, strongly stimulates the activity of the epithelial sodium channel (ENaC). Interestingly, other factors appear to regulate SGK1 gene expression and kinase activity. Insulin, for example, stimulates SGK1 activity (but not gene transcription) through its effects on phosphatidylinositol-3-kinase and osmotic shock appears to stimulate both SGK1 activity and gene transcription. Hence, SGK1 might integrate the effects of multiple hormonal and nonhormonal regulators of Na+ transport in tight epithelia and thereby play a key role in volume homeostasis. It is interesting to speculate that SGK1 might be implicated in medical conditions, such as the insulin resistance syndrome, hypertension and congestive heart failure.
David Pearce Division of Nephrology, Dept of Medicine and Dept of Cellular & Molecular Pharmacology, Box 0532, University of California, San Francisco, San Francisco, CA 94143, USA. e-mail: pearced@ medicine.ucsf.edu
Aldosterone is one of the key regulators of blood pressure (BP) and electrolyte homeostasis in vertebrates, owing, in large measure, to its effects on Na+ transport in tight epithelia. It binds with high affinity to the mineralocorticoid receptor (MR) and with lower but significant affinity to the glucocorticoid receptor (GR), both of which are members of the steroid receptor family of hormonedependent transcription factors (reviewed in Refs 1–3). Although MR is the principal mediator of aldosterone-stimulated ion transport in epithelia, it appears that GR contributes when aldosterone concentrations are high4,5. Hormone binding triggers a conformational change in all steroid and nuclear receptors that results in release of inactivating chaperones, translocation to the nucleus and/or changes in subnuclear architecture and regulation of specific target genes6,7. A substantial amount of recent effort in the steroid- and nuclear-receptor fields has focused on the protein–DNA and protein–protein interactions that lead to the formation of transcription preinitiation complexes and productive gene transcription. Hence, many of the general features of steroid and nuclear receptor regulation of gene expression in general, and MR and GR function in particular, have been well characterized (reviewed in Refs 1,2,8). However, until recently, the specific target genes and mechanisms involved in mineralocorticoid-regulated ion transport http://tem.trends.com
in epithelia remained obscure. Considerable progress has now been made in delineating the molecular pathways that link receptor-mediated changes in gene transcription with changes in ion transport. This review will focus on the role of the serum and glucocorticoid-regulated kinase (SGK) family, particularly isoform 1 (SGK1), in mineralocorticoidregulated Na+ transport, and will discuss the possible role of SGK1 as an integrator of multiple signals. The identification of novel isoforms of SGK (SGK2 and SGK3) and the possible role of the SGK family in the insulin resistance syndrome also will be addressed. Early effects of aldosterone in tight epithelia result from changes in gene transcription of a Na+ channel regulator
The physiological mechanisms underpinning aldosterone action, particularly in the kidney collecting duct (CD), have been extensively characterized in whole animals, isolated tubules and in cultured cells (reviewed in Ref. 9). In intact CD or cultured CD cells, the response to mineralocorticoids can be divided into two main phases – early and late – that differ significantly, although both require changes in gene transcription (reviewed in Ref. 3). The principal early action of aldosterone in CD cells (starting after a latent period of 30–45 min) is to increase apical membrane permeability by increasing Na+ transport through the epithelial Na+ channel (ENaC)9,10. The ENaC is probably a heterotetramer composed of three different subunits (α, β and γ) in the ratio 2:1:1 (Ref. 11). It is expressed in a variety of Na+-transporting epithelia, including lung, colon and parotid gland, in addition to CD, where it constitutes the rate-limiting step in transepithelial Na+ transport. The biophysical and physiological properties of the ENaC have been discussed extensively in numerous reviews (e.g. Ref. 9). One aspect of its regulation that warrants particular comment in the present context is the regulatory action of the ubiquitin ligase, Nedd4 (neural precursor cell expressed, developmentally downregulated 4). Nedd4 was originally identified in a subtracted library representing genes downregulated during development of the mouse brain12. Although its function in neural development remains uncertain, it was subsequently independently cloned as an interacting protein for the C-terminal tail of the ENaCβ subunit13. Analysis of a series of truncations and point mutations revealed
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Fig. 1. Northern blot analysis revealing rapid and transient induction of SGK1 mRNA synthesis in A6 cells treated with dexamethasone. Blots were hybridized with a probe for Xenopus SGK1 and type 8 actin29. Dexamethasone is the most potent stimulus of SGK1 expression in A6 cells; aldosterone gives a similar but less robust effect. Reproduced, with permission, from Ref. 29.
that a tryptophan-rich region of Nedd4 (‘WW domain’) stably interacts with a proline- and tyrosinerich amino stretch in Nedd4 termed the ‘PY’ motif. Following Nedd4-dependent ubiquitination, channel proteins are removed from the plasma membrane and either recycled or undergo proteasome-mediated degradation. Interestingly, Liddle’s syndrome, a form of pseudoaldosteronism, results from ENaC mutations that disrupt the PY motif and interaction with Nedd4 (Refs 13–15). In most cases, the ENaC-mediated early effect of aldosterone accounts for more than 60% of the total increase in Na+ current3 and, importantly, occurs without changes in ENaC gene transcription or mRNA levels16–24. Since aldosterone action clearly requires changes in gene transcription, these observations suggested the hypothesis that a key mineralocorticoid-regulated gene (or genes) encoded a regulatory protein that increases the plasma membrane localization and/or activity of already synthesized ENaC subunits. Nevertheless, regulation of α-subunit biosynthesis might be important for the late effects of aldosterone20,24. These considerations prompted several groups to search for ENaC regulators as mediators of the early aldosterone effect. Many candidates, however, had pleiotrophic effects or were only modestly or slowly stimulated9,25–28. The early effects of aldosterone on Na+ transport are potent, rapid and largely limited to changes in ENaC-mediated Na+ transport; other effects, such as changes in the proliferative state of CD cells or increases in membrane surface area, occur later29. Thus, the mRNA and protein levels of a mediator of the early response should increase markedly and rapidly and the mediator should, in turn, strongly stimulate ENaC activity. Recently, SGK1 has been shown to be just such an aldosteronestimulated regulator of ENaC activity30–33. SGK1 mRNA is rapidly increased by mineralocorticoids and, when expressed in Xenopus oocytes, SGK1 strongly and selectively stimulates ENaC-mediated Na+ transport in addition to the localization of ENaC in the membrane. Interestingly, SGK1 activity (but not abundance) is regulated by insulin, suggesting http://tem.trends.com
SGK1 was originally identified as a glucocorticoidregulated mRNA in mammary epithelial cells and termed SGK to reflect its regulation at the transcriptional level by serum and glucocorticoids34. It was subsequently cloned by several labs and its expression level was found to be regulated by a variety of hormonal and non-hormonal stimuli, including serum, follicle stimulating hormone (FSH)35, osmotic shock36 and mineralocorticoids30–32. It has the characteristic motifs of a serine/threonine kinase and kinase activity has been demonstrated in vitro37–39. SGK1 is found in most vertebrate tissues, although at variable levels34, and its expression pattern within tissues shows considerable heterogeneity with respect to both basal and stimulated levels of expression31. Thus, in kidney CD, basal levels of SGK1 are low and are strongly induced by mineralocorticoids, whereas in proximal tubule, expression is undetectable (by in situ hybridization) in the absence or presence of mineralocorticoids or glucocorticoids. In glomerular mesangial cells, moderate basal levels of SGK1 are detected that appear to be uninfluenced by corticosteroids. Interestingly, TGFβ (transforming growth factor β) could be a potent stimulator of SGK1 expression in this cell type40. The regulation of SGK1 expression in other tissues is similarly cell-type specific36,41. SGK1 mRNA and protein levels are regulated by mineralocorticoids in CD and colon
In an attempt to identify candidate mineralocorticoid response genes involved in the control of Na+ transport, our lab used suppression subtractive hybridization to generate a cDNA library enriched in dexamethasone-induced gene products in A6 cells42. From the resulting library, one clone showed very high homology to rat and human SGK1 and was selected for further characterization. Northern blot analysis confirmed rapid and transient dexamethasone induction of SGK1 mRNA in A6 cells (Fig. 1), and not surprisingly, induction of SGK1 mRNA was not blocked by cycloheximide30, suggesting that it has a direct transcriptional effect. Although dexamethasone was used to generate the library because of the substantially higher levels of GR than MR in these cells43,44, high concentrations of aldosterone, sufficient to activate GR, similarly stimulated SGK1 gene expression (unpublished). It is important to note that ‘glucocorticoids’ can have ‘mineralocorticoid’ effects in classical mineralocorticoid target cells45: their failure to act as mineralocorticoids does not depend on receptor specificity, but rather on the action of an enzyme, 11β-hydroxysteroid dehydrogenase46. Because this
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Fig. 2. In situ hybridization of SGK1 in rat kidney. Adrenalectomized rats were treated for 4 h with (a) vehicle and then hybridized with sense SGK1 mRNA, (b) vehicle and then hybridized with anti-sense SGK1 mRNA and (c) vehicle + aldosterone and hybridized with anti-sense SGK1 mRNA (Ref. 29). Note the appearance of the cortex in (c), suggesting ‘medullary rays’. Inspection of emulsion-dipped sections confirmed that these are indeed cortical collecting ducts. Abbreviation: SGK1, serum and glucocorticoid-regulated kinase isoform 1. Reproduced, with permission, from Ref. 29.
issue continues to cause confusion, many investigators use superphysiological doses of aldosterone to stimulate Na+ transport and infer that the effect is more reflective of true mineralocorticoid physiology than if they used dexamethasone or another ‘glucocorticoid’. For reasons that remain uncertain, most stable cell lines lose MR expression5,43,44,47. In these cells, the effects of aldosterone are mediated primarily, if not solely, by GR (hence the high concentrations of aldosterone required to stimulate Na+ currents)4. In CD cells and possibly other Na+-transporting epithelia that express both MR and GR, both receptors mediate similar, if not identical effects on gene transcription and on ion transport44,45. Hence, the term ‘mineralocorticoid’ (defined functionally) is appropriate for dexamethasone in this context. In contexts where MR and GR mediate strikingly different effects, the distinction is mechanistically extremely important49,50. It seems likely that the semantic confusion that still surrounds this topic will become clearer as the mechanisms underlying mineralocorticoid action are fully elucidated. Regardless of terminology, conclusions drawn from artificial systems must be verified in systems that are (a)
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Fig. 3. SGK1 stimulates ENaC but not ROMK2 activity. (a) In vitro transcribed RNA for each of the three ENaC subunits was injected into Xenopus laevis oocytes with or without SGK1. Shown is amiloride-sensitive current (Iami). (b) In vitro transcribed ROMK2 mRNA was injected into Xenopus oocytes with or without SGK1, as in (a). ROMK2 is the principal potassium channel found in collecting duct apical membrane. Abbreviations: ENaC, epithelial Na+ channel; ROMK2, principal collecting duct K+ channel; SGK1, serum and glucocorticoid-regulated kinase isoform 1. Reproduced, with permission, from Ref. 29.
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of clear physiological relevance. Importantly, aldosterone has been shown to stimulate SGK1 expression in both the CD of intact animals30,50, and in primary cultures of CD cells31, which express both MR and GR at high levels45. Our lab used in situ hybridization to show that SGK1 is induced by aldosterone in the CD of rat kidney (Fig. 2). The response of SGK1 to the physiological mineralocorticoid aldosterone was rapid, beginning within 0.5 h of a single injection of aldosterone; its levels also decreased rapidly, returning to near baseline by 24 h (Refs 30,50; unpublished), consistent with the short half-life of both aldosterone and SGK1 (Ref. 51). Both the time course and dose response of SGK1 to aldosterone correlate well with aldosteroneinduced changes in Na+ and K+ transport50. SGK1 stimulates ENaC-mediated Na+ transport and plasma membrane localization
ENaC constitutes the rate-limiting step for Na+ transport in aldosterone-responsive epithelia and is the principal early target of aldosterone action9,18. Consistent with an important role in mediating the early effects of aldosterone, SGK1 strongly stimulates ENaC-mediated Na+ currents in a Xenopus oocyte coexpression assay30–33 (Fig. 3). Importantly, as noted above, a kinase-dead mutant of SGK1 does not stimulate ENaC and indeed, has dominant negative activity52. Furthermore, SGK1 does not stimulate principal collecting duct K+ channel (ROMK2)mediated K+ transport, an observation that is of interest, not only because it suggests specificity to the SGK1 effect, but also because ROMK2 is the major pathway for K+ transport in CD. This observation is consistent with the view that changes in K+ transport depend on alterations in the electrochemical gradient established by Na+ movement through the ENaC, although additional regulators could be implicated. Interestingly, recent evidence supports the possibility that SGK1 does stimulate a K+ channel unrelated to ROMK2, Kv1.3*. The significance of this observation, however, remains to be determined. Initial insight into the mechanism of SGK1 stimulation of ENaC is provided by the observation that the kinase increases ENaC plasma membrane localization to an extent comparable with its stimulation of Na+ current, when expressed in Xenopus oocytes52,53 (Fig. 4). This observation is particularly relevant in the light of mounting evidence that aldosterone stimulates ENaC-mediated Na+ transport by increasing its apical membrane localization24,52. Importantly, SGK1 kinase activity is required, both for stimulation of Na+ current, and for increased ENaC plasma membrane localization50 (Fig. 4). Indeed, kinase-dead forms of SGK1 have dominant negative activity, probably because they *Beck, S. et al. (2001) The aldosterone-regulated serine/threonine kinase SGK1 stimulates the Kv1.3 potassium channel. Joint Meeting of the Scandinavian Physiological Society and of the German Physiological Society
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displace wild-type SGK1 from cellular targets. In contrast to its effect on plasma membrane localization, SGK1 appears to have little or no effect on open probability of the channels53; however, this issue remains unsettled and it is possible that under physiological conditions, SGK1 could impact on open probability, as has been suggested for aldosterone16.
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Fig. 4. Wild-type (wt) SGK1 stimulates ENaC-mediated current (a) and ENaC surface localization (b,c) in Xenopus laevis oocytes. A kinase-dead mutant (mut SGK) has the opposite effect50. (a) Either the wild type or K130A mutant (unable to bind ATP) of SGK1 was coexpressed with ENaC subunits in Xenopus oocytes and amiloride-sensitive current (Iami) was measured by two-electrode voltage clamp. (b) Surface expression of FLAG-epitope tagged ENaC subunits was detected by binding of radioiodinated anti-FLAG antibody. (c) FLAG–ENaC surface expression detected by immunostaining in (i) control, (ii) wt SGK and (iii) mut SGK oocytes. Abbreviations: ENaC, epithelial Na+ channel; SGK, serum and glucocorticoid-regulated kinase. Reproduced, with permission, from Ref. 50.
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Although aldosterone is the main physiological regulator of CD Na+ transport, insulin also stimulates reabsorption54–56, a process that might be of considerable relevance to the pathogenesis of hypertension associated with the insulin resistance syndrome (‘syndrome X’)57. Interestingly, the closest relative of SGK1 (other than SGK2 and 3) is protein kinase B (PKB)/Akt, a member of the serine/threonine kinase family, which plays a central role in insulin signaling and has been studied extensively58. PKB/Akt is activated by phosphatidylinositol-3-kinase (PI3K) through phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent kinase 1 (PDK1)59. SGKs of all species, including yeast, have PDK consensus sites like those of PKB/Akt; recent evidence indicates that they are phosphorylation targets of PDK1 (and possibly PDK2)38,60, and that their activities are PI3Kdependent37. Importantly, blockade of PI3K not only abolishes insulin-stimulated Na+ transport61 and the early component of mineralocorticoid-stimulated Na+ transport, but also eliminates phosphorylation of SGK162,63 (Fig. 5a,b). Furthermore, insulin, which has no effect on SGK1 gene expression, augments SGK1 activity by increasing its phosphorylation37,60,63 (Fig. 5c). Under these conditions, mineralocorticoids and insulin have been found to stimulate Na+ transport in CD cells synergistically63–65 (Fig. 5c). These observations suggest that SGK1 is an integrator of insulin and mineralocorticoids in the control of Na+ transport, as shown schematically in Fig. 6. However, aldosterone–insulin synergy has not always been found66.
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Fig. 5. (a) Amiloride-inhibitable equivalent current, which represents ENaC-mediated Na+ transport, showing blocking of the early mineralocorticoid effect on Na+ transport by PI3K inhibition in A6 cells. Cells were treated with the specific PI3K inhibitor, LY294002 (circles), or nothing (squares), before addition of dexamethasone after 0.5 h. PI3K inhibitor + dexamethasone (triangles) and dexamethasone only (diamonds). Where not shown, error bars were smaller than data symbols. (b) Immunoblots probed for SGK1 at different times, demonstrating block of SGK1 phosphorylation by PI3K inhibition in A6 cells. Cells were treated (i) without and (ii) with PI3K inhibitor and the upper and lower bands are the phosphorylated and unphosphorylated forms of SGK1, respectively. (c) Amiloride-inhibitable equivalent current, showing synergy between insulin, which increases phosphorylation of SGK1, and dexamethasone in stimulating ENaC-mediated Na+ transport in A6 cells. Cells were treated with dexamethasone (or vehicle) alone for 2 h, followed by addition of insulin (or vehicle) for an additional 0.5 h and then harvested and immunoblotted for SGK1. Changes in equivalent current relative to that of epithelia untreated with dexamethasone or insulin were (in µA cm−2): 4.1 (dexamethasone alone); 5.8 (insulin alone); 15.3 (dexamethasone + insulin). The change in equivalent current induced by insulin and dexamethasone together was significantly greater than the sum of those induced by the two separately (15.3 versus 9.9 µA; P < 0.001, unpaired Student’s ttest). Ratio of phosphorylated to unphosphorylated SGK1 was significantly higher in the presence of insulin (P < 0.01, unpaired Student’s t-test), but was unaffected by dexamethasone. Abbreviations: Dex, dexamethasone; ENaC, epithelial Na+ channel; Ins, insulin; PI3K, phosphatidylinositol-3-kinase; SGK1, serum and glucocorticoid-regulated kinase isoform 1. Reproduced, with permission, from Ref. 29.
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SGK1 is a candidate integrator of insulin and aldosterone effects on Na+ transport
Other regulators of SGK1 activity and/or expression
A variety of other factors impact on SGK1 activity and/or expression. Osmotic shock, for example, potently stimulates SGK1 gene transcription36,67,68, whereas insulin stimulates its activity40 and FSH stimulates both35,69. Osmotic shock is a particularly intriguing stimulus of SGK1 expression in the present context because it is the most ancient effector of changes in cell volume, and an important determinant of the homeostatic response of freshwater and euryhaline vertebrates to changes in the ionic composition of their environment. In early (Precambrian) marine organisms, SGK1 might have been regulated by osmotic strength (possibly through changes in cell volume), with other regulators introduced later, most notably for our purposes, the renin–angiotensin–aldosterone system, which is first
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ROMK2
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necessary for growth factor-induced entry of cells into S-phase75. The intriguing possibility that SGK1induced changes in ion transport might be implicated in its antiapoptotic and/or proliferative effects remains to be examined.
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Fig. 6. A speculative scheme of hormone-regulated Na+ transport, emphasizing the role of SGK1. According to this scheme, SGK1 abundance is regulated by mineralocorticoids, whereas its activity is regulated by PI3K (through PIP3 and PDK1). SGK2 and SGK3 activities are thought to be similarly regulated through PI3K; however, their levels do not appear to be regulated by mineralocorticoids or glucocorticoids. Before its interaction with SGK1, ENaC is shown in a hypothetical vesicle, analogous to insulin-regulated vesicles. The dotted line between PIP3 and PDK1 indicates the stable, non-covalent interaction between PIP3 and the PH domain of PDK1. Abbreviations: ECF, extracellular fluid; ENaC, epithelial Na+ channel; Ins, insulin; IRS1, insulin receptor substrate 1; IRV, insulin-regulated vesicle; MC, mineralocorticoid; MR, mineralocorticoid receptor; PDK1, PIP3-dependent kinase 1; PH, pleckstrin homology domain; PI3K, phosphatidylinositol-3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; ROMK2, principal collecting duct K+ channel; SGK1, serum and glucocorticoidregulated kinase isoform 1. Reproduced, with permission, from Ref. 29.
seen in amphibia or possibly (in a rudimentary form) in bony fish70. A teleological reason for the regulation of SGK1 by other hormones such as insulin, TGFβ and FSH is less clear. This is, in part, because until recently, the only clearly established physiological role for SGK1 was in the control of epithelial ion transport71,72. However, recent evidence has begun to provide direct support for the idea that SGK1, in some settings, might be implicated in the control of proliferation and apoptosis, as suggested previously34. Thus, SGK1 was recently shown to phosphorylate and inactivate the Forkhead family member, FKHRL1, a proapoptotic transcription factor previously known to be a regulatory target of Akt/PKB (Refs 39,73). In addition to this powerful, albeit indirect, evidence, one recent report provided direct evidence that SGK1 can be antiapoptotic74. Furthermore, another recent report directly demonstrated that phosphorylation of SGK1 at Ser78 by the mitogen activated kinase BMK1 is http://tem.trends.com
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Other SGK isoforms
Recently, two additional isoforms of SGK were identified60. Hence, the originally identified, glucocorticoid–mineralocorticoid-regulated form of SGK is now referred to as SGK1. Neither of these novel isoforms is glucocorticoid-regulated, and preliminary data suggest that they are not mineralocorticoid regulated either (D. Pearce, unpublished). The three isoforms of SGK share significant nucleotide (~60%) and amino acid (~80%) identity in their kinase domains and potential for misidentification and functional cross talk exists. SGK3, like SGK1, is expressed in all tissues examined by northern blot, whereas SGK2 is expressed only in kidney, liver, pancreas and brain. The distribution of SGK2 and SGK3 within tissues has not been examined and their physiological roles are unknown. However, it has been established that both stimulate ENaC-mediated Na+ transport†. A mechanistic view of SGK-stimulated Na+ transport
SGK1 stimulation of ENaC-mediated Na+ transport clearly requires its kinase activity52 (Fig. 4). However, in spite of attempts by several investigators, phosphorylation of ENaC subunits by SGK1 has not been demonstrated51 (D. Pearce, unpublished; F. Lang, pers. commun.). Moreover, SGK1 and probably SGK2 and SGK3 increase ENaC localization to the plasma membrane, although an effect on channel open probability has not been ruled out. Hence, it seems likely that the target proteins of the SGKs are involved in ENaC translocation to the plasma membrane, its removal, or both. Although there is some evidence suggesting that ENaC removal is not reduced by SGK1 (Ref. 53), additional evidence is needed to settle this issue. In principle, the most efficient way to achieve the rapid and potent effects that aldosterone and SGK1 have on ENaC is to alter both insertion and removal of channels. With regard to regulation of channel insertion, striking parallels between glucose transporter (GLUT4)-mediated glucose transport and ENaC-mediated Na+ transport have become apparent, perhaps reflecting common themes that underlie the hormonal control of protein trafficking events via intracellular signaling kinases76,77. Although the principal PKB/Akt targets involved in the exocytotic process have not been identified, recently, Synip, an insulin-regulated syntaxin 4-interacting protein, was identified as a potential regulator of GLUT4 vesicle fusion78. †Friederich, B. et al. (2000) Serine/Threonine kinases SGK2 and SGK3 both upregulate renal epithelial Na+-channel α, β, γ ENaC. American Society of Nephrology Annual Meeting Abstr. 149
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Acknowledgements Work performed in the author’s laboratory was supported by NIH grants DK51151 and DK56695 as well as a Grant-in-Aid from the American Heart Association Western States Affiliate. Gary Firestone is gratefully acknowledged for suggestions on the manuscript. Florian Lang and Olivier Staub are gratefully acknowledged for communicating unpublished data.
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With regard to mineralocorticoid regulation of channel removal, it is interesting to return to Liddle’s syndrome, which probably results from disruption of ENaC interaction with Nedd4 (Refs 13,14,79). In light of the opposing actions of SGK and Nedd4 on ENaC-mediated Na+ transport and the clinical similarities between Liddle’s syndrome and primary aldosteronism, it is plausible that SGK1 might influence Nedd4 activity, or vice versa. Importantly, since aldosterone does not regulate Nedd4 gene transcription80, any role that Nedd4 plays in aldosterone action must be indirect or as a target of an aldosterone-regulated gene product, such as SGK. According to the speculative scheme shown in Fig. 6, both phosphorylated and unphosphorylated forms of SGK1 are recruited to ENaC-containing vesicles by the channel itself, as suggested by recent evidence that SGK1 physically interacts with ENaC subunits, in vitro63. The activated form of SGK1 (phosphorylated at Ser256 and Ser422; human numbering) then phosphorylates proteins (possibly Synip or a Synip-like protein) that regulate channel
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insertion (via vesicle fusion with the plasma membrane) and possibly removal (for example Nedd4). The resulting increase in ENaC present at the apical membrane largely accounts for the increase in Na+ transport, although coincident changes in channel-open probability might also occur. This scheme accounts for the dominant negative activity of kinase-dead forms of SGK1, which interact with ENaC nonproductively, displacing wild-type kinase52. The dual regulation of SGK1 – of its abundance through a transcriptional mechanism and of its activity through a PI3K-dependent pathway – provides a mechanism for integrating multiple different inputs that impact on Na+ influx into epithelial cells, particularly those of aldosterone and insulin63. It will be of considerable interest for future investigation to determine if SGK1 plays a role in the salt-sensitive hypertension associated with the insulin-resistance syndrome57. Furthermore, the roles of SGK2 and SGK3 in normal signaling and disease pathogenesis remain to be characterized.
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