Guido Henke

The Serum and Glucocorticoid-Inducible Kinase SGK1 and the Na+/H+ Exchange Regulating Factor NHERF2 Synergize to Stimulate the Renal Outer Medullary K+ Channel ROMK1

Mineralocorticoids stimulate Na(+) reabsorption and K(+) secretion in principal cells of connecting tubule and collecting duct. The involved ion channels are ENaC and ROMK1, respectively. In Xenopus oocytes, the serum and glucocorticoid-sensitive kinase SGK1 has been shown to increase ENaC activity by enhancing its abundance in the plasma membrane. With the same method, ROMK1 appeared to be insensitive to regulation by SGK1. On the other hand, ROMK1 has been shown to colocalize with NHERF2, a protein mediating targeting and trafficking of transport proteins into the cell membrane. The present study has been performed to test whether NHERF2 is required for regulation of ROMK1 by SGK1. Coexpression of neither NHERF2 nor SGK1 with ROMK1 increases ROMK1 activity. However, coexpression of NHERF2 and SGK1 together with ROMK1 markedly increases K(+) channel activity. The combined effect of SGK1 and NHERF2 does not significantly alter the I/V relation of the channel but increases the abundance of the channel in the membrane and decreases the decay of channel activity after inhibition of vesicle insertion with brefeldin. Coexpression of NHERF2 and SGK1 does not modify cytosolic pH but leads to a slight shift of pK(a) of ROMK1 to more acidic values. In conclusion, NHERF2 and SGK1 interact to enhance ROMK1 activity in large part by enhancing the abundance of channel protein within the cell membrane. This interaction allows the integration of genomic regulation and activation of SGK1 and NHERF2 in the control of ROMK1 activity and renal K(+) excretion.

Regulation of Channels by the Serum and Glucocorticoid-Inducible Kinase - Implications for Transport, Excitability and Cell Proliferation

The serum and glucocorticoid-inducible kinase SGK1 stimulates the Na+ channels ENaC and SCN5A, the K+ channels ROMK1, Kv1.3, and KCNE1/KCNQ1, the cation conductance induced by 4F2/LAT1 and the chloride conductance induced by CFTR. The isoforms SGK2 and SGK3 have similarly been shown to regulate ENaC, SCN5A, Kv1.3 and KCNE1/KCNQ1. The kinases regulate channel abundance in the plasma membrane in part by inhibition of the ubiquitin ligase Nedd4-2 and in part by interaction with trafficking molecules such as the Na+/H+ exchanger regulating factor NHERF2. An in vivo role of SGK1 mediated ENaC channel regulation in renal salt excretion and blood pressure control is documented by the impaired ability of SGK1 knockout mice to adequately reduce renal Na+ output and maintain blood pressure during dietary salt restriction and by enhanced blood pressure in individuals carrying certain polymorphisms in the SGK1 gene. The in vivo physiological significance of SGK dependent regulation of the other channels remains to be shown even though circumstantial evidence points to involvement in the regulation of epithelial transport, cell volume, cell proliferation, cardiac action potential and neuroexcitability. There is little doubt that further channels will be identified which are modulated by the SGKs and that further in vivo physiological functions will be defined where channel regulation by the SGKs plays a critical role.

Regulation of the glutamate transporter EAAT1 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid-inducible kinase isoforms SGK1/3 and protein kinase B

Surface expression of the glial glutamate transporter EAAT1 is stimulated by insulin‐like growth factor 1 through activation of phosphatidylinositol‐3‐kinase. Downstream targets include serum and glucocorticoid‐sensitive kinase isoforms SGK1, SGK2 and SGK3, and protein kinase B. SGK1 regulates Nedd4‐2, a ubiquitin ligase that prepares cell membrane proteins for degradation. To test whether Nedd4‐2, SGK1, SGK3 and protein kinase B regulate EAAT1, cRNA encoding EAAT1 was injected into Xenopus oocytes with or without additional injection of wild‐type Nedd4‐2, constitutively active S422DSGK1, inactive K127NSGK1, wild‐type SGK3 and/or constitutively active T308D,S473DPKB. Glutamate induces a current in Xenopus oocytes expressing EAAT1, but not in water‐injected oocytes, which is decreased by co‐expression of Nedd4‐2, an effect reversed by additional co‐expression of S422DSGK1, SGK3 and T308D,S473DPKB, but not K127NSGK1. Site‐directed mutagenesis of the SGK1 phosphorylation sites in the Nedd4‐2 protein (S382A,S468ANedd4‐2) and in the EAAT1 protein (T482AEAAT1, T482DEAAT1) significantly blunts the effect of S422DSGK1. Moreover, the current is significantly larger in T482DEAAT1‐ than in T482AEAAT1‐expressing oocytes, indicating that a negative charge mimicking phosphorylation at T482 increases transport. The experiments reveal a powerful novel mechanism that regulates the activity of EAAT1. This mechanism might participate in the regulation of neuronal excitability and glutamate transport in other tissues.

Regulation of the voltage gated K+ channel Kv1.3 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid inducible kinase SGK1

The stimulation of cell proliferation by insulin like growth factor IGF‐1 has previously been shown to depend on activation of voltage gated K+ channels. The signaling involved in activation of voltage gated K+ channel Kv1.3 includes the phosphatidylinositol‐3 (PI3) protein kinase, 3‐phosphoinositide dependent protein kinase PDK1 and the serum and glucocorticoid inducible kinase SGK1. However, nothing is known about mechanisms mediating the stimulation of Kv1.3 by SGK1. Most recently, SGK1 has been shown to phosphorylate and thus inactivate the ubiquitin ligase Nedd4‐2. The present study has been performed to explore whether the regulation of Kv1.3 involves Nedd4‐2. To this end Kv1.3 has been expressed in Xenopus oocytes with or without coexpression of Nedd4‐2 and/or constitutively active S422DSGK1. In oocytes expressing Kv1.3 but not in water injected oocytes, depolarization from a holding potential of −80 mV to +20 mV triggers rapidly inactivating currents typical for Kv1.3. Coexpression of Nedd4‐2 decreases, coexpression of S422DSGK1 enhances the currents significantly. The effects of either Nedd4‐2 or of SGK1 are abrogated by destruction of the respective catalytic subunits (C938SNedd4‐2 or K127NSGK1). Further experiments revealed that wild type SGK1 and SGK3 and to a lesser extent SGK2 are similarly effective in stimulating Kv1.3 in both, presence and absence of Nedd4‐2. It is concluded that Kv1.3 is downregulated by Nedd4‐2 and stimulates by SGK1, SGK2, and SGK3. The data thus disclose a novel mechanism of Kv1.3 channel regulation. J. Cell. Physiol. 199: 194–199, 2004© 2003 Wiley‐Liss, Inc.

Stimulation of Xenopus oocyte Na+,K+ATPase by the serum and glucocorticoid-dependent kinase sgk1

Abstract. The serum and glucocorticoid-dependent kinase-1 (sgk1) is expressed in a wide variety of tissues including renal epithelial cells. As it is up-regulated by aldosterone, it is considered to participate in the regulation of renal Na+ reabsorption. Indeed, co-expression of sgk1 with the renal epithelial Na+ channel (ENaC) augments Na+ channel activity. The aim of the present study was to examine possible effects of sgk1 on Na+/K+-ATPase activity. To this end dual-electrode voltage-clamp experiments were performed in Xenopus oocytes expressing the active kinase S422Dsgk1 or the inactive mutant K127Nsgk1. Na+/K+-ATPase activity was estimated from the hyperpolarization (ΔVm) and the outwardly-directed current (IP) created by addition of extracellular K+ in the presence of K+ channel blocker Ba2+. Both ΔVm and IP were significantly larger in oocytes expressing S422Dsgk1 than in those expressing K127Nsgk1 or having been injected with water. IP was fully inhibited by ouabain. Ion-selective microelectrodes showed that the stimulation of pump current was not the result of altered cytosolic Na+ activity or pH. The present results thus point to an additional action of sgk1 that may participate in the regulation of renal tubular Na+ transport. Moreover, sgk1 may be involved in the regulation of Na+/K+-ATPase in extrarenal tissues.

Serum and glucocorticoid inducible kinases in the regulation of the cardiac sodium channel SCN5A

The serum and glucocorticoid inducible kinase SGK1 and its isoform SGK3 are both expressed in cardiac tissue. One of the functions of SGK1 is the phosphorylation and inactivation of the ubiquitin ligase Nedd4-2, which in turn could be shown to downregulate the voltage-gated Na+ channel SCN5A (hH1). The present study has been performed to test for a role of SGK1 and SGK3 in the regulation of SCN5A. To this end cRNA encoding the human Na+ channel SCN5A was injected into Xenopus laevis oocytes with or without cRNA encoding the wild-type kinases SGK1, the constitutively active kinase (S422D)SGK1, the inactive form K127NSGK1 or the wild-type SGK3. SCN5A currents were activated by coexpression of either wild-type SGK1 or SGK3 or the constitutively active S422DSGK1. In contrast, the inactive mutant K127NSGK1 significantly decreased the currents. Moreover, coexpression of SGK3 significantly altered SCN5A gating, i.e. it hyperpolarized the activation threshold and depolarized the prepotential required for 50% availability of the channel. Opposite shifts of gating properties were elicited by mutation of serine to alanine (S483ASCN5A and S663ASCN5A) in the SGK consensus sequences of SCN5A. The present observations disclose a role of the kinases SGK1 and SGK3 in the regulation of cardiac Na+ channels. As SGK1 is upregulated by glucocorticoids, mineralocorticoids and a variety of inflammatory mediators and both kinases are activated by insulin and IGF1, the kinases could mediate effects of those hormones and mediators on cardiac function.

Cerebral localization and regulation of the cell volume-sensitive serum- and glucocorticoid-dependent kinase SGK1

The serum- and glucocorticoid-dependent kinase SGK1 is regulated by alterations of cell volume, whereby cell shrinkage increases and cell swelling decreases the transcription, expression and activity of SGK1. The kinase is expressed in all human tissues studied including the brain. The present study was performed to localize the sites of SGK1 transcription in the brain, to elucidate the influence of the hydration status on SGK1 transcription and to explore the functional significance of altered SGK1 expression. Northern blot analysis of human brain showed SGK1 to be expressed in all cerebral structures examined: amygdala, caudate nucleus, corpus callosum, hippocampus, substantia nigra, subthalamic nucleus and thalamus. In situ hybridization and immunohistochemistry in the rat revealed increased expression of SGK1 in neurons of the hippocampal area CA3 after dehydration, compared with similar slices from brains of euvolaemic rats. Additionally, several oligodendrocytes, a few microglial cells, but no astrocytes, were positive for SGK1. The abundance of SGK1 mRNA in the temporal lobe, including hippocampus, was increased by dehydration and SGK1 transcription in neuroblastoma cells was stimulated by an increase of extracellular osmolarity. Co-expression studies in Xenopus laevis oocytes revealed that SGK1 markedly increased the activity of the neuronal K+ channel Kv1.3. As activation of K+ channels modifies excitation of neuronal cells, SGK1 may participate in the regulation of neuronal excitability.

Hypofractionated reirradiation for recurrent malignant glioma.

Background and Purpose:Treatment options for recurrent high-grade glioma after a complete course of radiotherapy comprise surgery, reirradiation and chemotherapy but the efficacy of any of the given salvage treatments is limited. In order to further define the role of short-term radiotherapy as retreatment option for selected patients, we analyzed outcomes after treatment with a hypofractionated radiation.Patients and Methods:Treatment outcomes (overall survival and treatment-associated toxicity) were analyzed retrospectively in 31 patients treated between 1994 and 2007. Hypofractionated radiotherapy was performed after three-dimensional CT planning with a median total dose of 20 Gy in a single department.Results:With a median interval of 20 months from primary radiotherapy, two grade III and 29 grade IV tumors were reirradiated. Pretreatment consisted of surgery and involved-field radiotherapy (median 59 Gy). 77% of the patients received additional chemotherapy before the second course of radiotherapy, and 48% were treated after secondary resection. The median overall survival after hypofractionated radiotherapy was 10.2 months, and the median overall survival time after primary diagnosis 30.9 months. No severe toxicity was observed.Conclusion:Hypofractionated reirradiation with 20 Gy given over 1 week is a practicable and well-tolerated treatment option for patients with recurrent malignant glioma. The overall survival was comparable to the reported outcomes from other series including those with longer treatment protocols.Hintergrund und Ziel:Die aktuellen Behandlungsoptionen bei Rezidiv eines vorbestrahlten hochmalignen Glioms beinhalten Operation, Strahlentherapie und Chemotherapie. Allerdings ist die Wirksamkeit der gegebenen Salvage-Behandlungen begrenzt. Um die Rolle einer kurzzeitigen Rebestrahlung bei ausgewählten Patienten weiter zu definieren, wurden Krankheitsverläufe bei Patienten nach Behandlung mit einem kurzzeitigen hypofraktionierten Bestrahlungskonzept analysiert.Patienten und Methodik:Die Verläufe (Gesamtüberleben und Toxizität) von 31 zwischen 1994 und 2007 behandelten Patienten wurden retrospektiv analysiert. Die hypofraktionierte Strahlentherapie wurde nach dreidimensionaler CT-Planung mit einer mittleren Gesamtdosis von 20 Gy in einer einzelnen Abteilung durchgeführt.Ergebnisse:Bei zwei Grad-III- und 29 Grad-IV-Tumoren wurde mit einem mittleren Abstand von 20 Monaten nach der primären Strahlentherapie eine Rebestrahlung durchgeführt. Die Vorbehandlung bestand in Operation und Involved-Field-Strahlentherapie (Median 59 Gy). 77% der Patienten erhielten vor dem zweiten Strahlentherapiekurs zusätzlich eine Chemotherapie, und 48% wurden nach erneuter Resektion behandelt. Das mittlere Gesamtüberleben nach hypofraktionierter Strahlentherapie betrug 10,2 Monate und das mittlere Gesamtüberleben nach Primärdiagnose 30,9 Monate. Schwere Toxizitäten wurden nicht beobachtet.Schlussfolgerung:Die hypofraktionierte Rebestrahlung mit 20 Gy über eine Woche ist eine praktikable und gut verträgliche Behandlungsform für Patienten mit Rezidiv eines hochgradigen malignen Glioms. Das Gesamtüberleben war mit den in der Literatur berichteten Resultaten anderer Serien einschließlich jener mit längeren Bestrahlungsprotokollen vergleichbar.

Activation of Na+/K+-ATPase by the Serum and Glucocorticoid-Dependent Kinase Isoforms

Background/Aim: Expression of the constitutively active form of serum and glucocorticoid-dependent kinase (^S422DSGK1) in Xenopus oocytes has recently been shown to upregulate endogenous Na⁺/K⁺-ATPase activity, an effect presumably participating in the regulation of cellular K⁺ uptake and transepithelial Na⁺ transport. SGK1 and the two isoforms SGK2 and SGK3 are stimulated by insulin and insulin-like growth factor-1 (IGF-1), which have been shown to enhance Na⁺/K⁺-ATPase activity in a variety of cells. The present experiments have been performed to elucidate whether or not wild-type SGK1, SGK2 and SGK3 are similar to ^S422DSGK1 in being effective regulators of Na⁺/K⁺-ATPase. Methods: To this end, dual-electrode voltage clamp experiments were performed in Xenopus oocytes injected either with water or with mRNA of constitutively active ^S422DSGK1 and wild-type SGK1, SGK2 or SGK3. Na⁺/K⁺-ATPase activity was estimated from the outward-directed current created by readdition of extracellular K⁺ in the presence of K⁺ channel blocker Ba²⁺ following a 10-min exposure to K⁺-free extracellular fluid. Results: The outward-directed current was fully abolished by incubation with 1 mM ouabain and was significantly larger in oocytes expressing ^S422DSGK1, SGK1, SGK2 or SGK3, as compared to those injected with water. Conclusion: The stimulating effect of SGK1 on the Xenopus oocyte Na⁺/K⁺-ATPase is mimicked by the isoforms SGK2 and SGK3. Thus, all three kinases may participate in the regulation of Na⁺/K⁺-ATPase activity by hormones such as insulin and IGF-1.

Simultaneous 68Ga-DOTATOC-PET/MRI for IMRT treatment planning for meningioma: first experience.

PURPOSE To evaluate intensity-modulated radiotherapy (IMRT) treatment planning based on simultaneous positron-emission tomography and magnetic resonance imaging (PET/MRI) of meningioma. METHODS AND MATERIALS A meningioma patient was examined prior to radiotherapy with dedicated planning computed tomography (CT), MRI, PET/CT with gallium-68-labeled DOTATOC (68Ga-DOTATOC), and simultaneous 68Ga-DOTATOC-PET/MRI. The first gross target volume (GTV) was defined based on a combination of separate MR and 68Ga-DOTATOC-PET/CT imaging (GTVPET/CT+MR). Then, the simultaneous PET/MR images were used to delineate a second GTV (GTVPET/MR) by following exactly the same delineation strategy. After an isotropic expansion of those volumes by a 4-mm safety margin, the resulting planning target volumes (PTVs) were compared by calculating the intersection volume and the relative complements. A cross-evaluation of IMRT plans was performed, where the treatment plan created for the PTVPET/CT+MR was applied to the PET/MR-based PTVPET/MR. RESULTS Generally, target volumes for IMRT treatment planning did not differ between MRI plus 68Ga-DOTATOC-PET/CT and simultaneous PET/MR imaging. Only in certain regions of the GTV were differences observed. The overall volume of the PET/MR-based PTV was approximately the same as that obtained from PET/CT data. A small region of infiltrative tumor growth next to the main tumor mass was better visualized with combined PET/MR due to smaller PET voxel sizes and improved recovery. An IMRT treatment plan was optimized for the PTVPET/CT+MR. The evaluation of this plan with respect to the PTVPET/MR showed parts of the target volume that would not have received the full radiation dose after delineation of the tumor, based on simultaneous PET/MR. CONCLUSION This case showed that differences in target volumes delineated on the basis of separate MR and PET/CT and simultaneous PET/MR may be observed that can have significant consequences for an effectively applied radiotherapy treatment plan.