Christina Leibrock

The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes

Platelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+](i) increase are significantly blunted in platelets from SGK1 knockout mice (sgk1(-/-)). Similarly, Ca2+ -dependent degranulation, integrin α(IIb)β3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1(-/-) platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1(-/-) mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1μM). Transfection of MEG-01 cells with (S422D)SGK1 significantly increased phosphorylation of IκB kinase α/β and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of (S422D)SGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10μM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB-dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice

Klotho is a potent regulator of 1,25-hydroxyvitamin D3 [1,25(OH)2D3] formation and calcium-phosphate metabolism. Klotho-hypomorphic mice (kl/kl mice) suffer from severe growth deficits, rapid aging, hyperphosphatemia, hyperaldosteronism, and extensive vascular and soft tissue calcification. Sequelae of klotho deficiency are similar to those of end-stage renal disease. We show here that the mineralocorticoid receptor antagonist spironolactone reduced vascular and soft tissue calcification and increased the life span of kl/kl mice, without significant effects on 1,25(OH)2D3, FGF23, calcium, and phosphate plasma concentrations. Spironolactone also reduced the expression of osteoinductive Pit1 and Tnfa mRNA, osteogenic transcription factors, and alkaline phosphatase (Alpl) in calcified tissues of kl/kl mice. In human aortic smooth muscle cells (HAoSMCs), aldosterone dose-dependently increased PIT1 mRNA expression, an effect paralleled by increased expression of osteogenic transcription factors and enhanced ALP activity. The effects of aldosterone were reversed by both spironolactone treatment and PIT1 silencing and were mitigated by FGF23 cotreatment in HAoSMCs. In conclusion, aldosterone contributes to vascular and soft tissue calcification, an effect due, at least in part, to stimulation of spironolactone-sensitive, PIT1-dependent osteoinductive signaling.

Hyperaldosteronism in Klotho-deficient mice

Klotho is a membrane protein participating in the inhibitory effect of FGF23 on the formation of 1,25-dihydroxyvitamin-D(3) [1,25(OH)(2)D(3)]. It participates in the regulation of renal tubular phosphate reabsorption and stimulates renal tubular Ca(2+) reabsorption. Klotho hypomorphic mice (klotho(hm)) suffer from severe growth deficit, rapid aging, and early death, events largely reversed by a vitamin D-deficient diet. The present study explored the role of Klotho deficiency in mineral and electrolyte metabolism. To this end, klotho(hm) mice and wild-type mice (klotho(+/+)) were subjected to a normal (D(+)) or vitamin D-deficient (D(-)) diet or to a vitamin D-deficient diet for 4 wk and then to a normal diet (D(-/+)). At the age of 8 wk, body weight was significantly lower in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice, klotho(hm)D(-) mice, and klotho(hm)D(-/+) mice. Plasma concentrations of 1,25(OH)(2)D(3,) adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), and aldosterone were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. Plasma volume was significantly smaller in klotho(hm)D(-/+) mice, and plasma urea, Ca(2+), phosphate and Na(+), but not K(+) concentrations were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. The differences were partially abrogated by a vitamin D-deficient diet. Moreover, the hyperaldosteronism was partially reversed by Ca(2+)-deficient diet. Ussing chamber experiments revealed a marked increase in amiloride-sensitive current across the colonic epithelium, pointing to enhanced epithelial sodium channel (ENaC) activity. A salt-deficient diet tended to decrease and a salt-rich diet significantly increased the life span of klotho(hm)D(+) mice. In conclusion, the present observation disclose that the excessive formation of 1,25(OH)(2)D(3) in Klotho-deficient mice results in extracellular volume depletion, which significantly contributes to the shortening of life span.

SGK3 regulates Ca(2+) entry and migration of dendritic cells.

Background/Aims: Dendritic cells (DCs) are antigen-presenting cells linking innate and adaptive immunity. DC maturation and migration are governed by alterations of cytosolic Ca²⁺ concentrations ([Ca²⁺]_i). Ca²⁺ entry is in part accomplished by store-operated Ca²⁺ (SOC) channels consisting of the membrane pore-forming subunit Orai and the ER Ca²⁺ sensing subunit STIM. Moreover, DC functions are under powerful regulation of the phosphatidylinositol-3-kinase (PI3K) pathway, which suppresses proinflammatory cytokine production but supports DC migration. Downstream targets of PI3K include serum- and glucocorticoid-inducible kinase isoform SGK3. The present study explored, whether SGK3 participates in the regulation of [Ca²⁺]_i and Ca²⁺-dependent functions of DCs, such as maturation and migration. Methods/ Results: Experiments were performed with bone marrow derived DCs from gene targeted mice lacking SGK3 (sgk3^-/-) and DCs from their wild type littermates (sgk3^+/+). Maturation, phagocytosis and cytokine production were similar in sgk3^-/- and sgk3^+/+ DCs. However, SOC entry triggered by intracellular Ca²⁺ store depletion with the endosomal Ca²⁺ ATPase inhibitor thapsigargin (1 µM) was significantly reduced in sgk3^-/- compared to sgk3^+/+ DCs. Similarly, bacterial lipopolysaccharide (LPS, 1 µg/ml)- and chemokine CXCL12 (300 ng/ml)- induced increase in [Ca²⁺]_i was impaired in sgk3^-/- DCs. Moreover, currents through SOC channels were reduced in sgk3^-/- DCs. STIM2 transcript levels and protein abundance were significantly lower in sgk3^-/- DCs than in sgk3^+/+ DCs, whereas Orai1, Orai2, STIM1 and TRPC1 transcript levels and/or protein abundance were similar in sgk3-/- and sgk3^+/+ DCs. Migration of both, immature DCs towards CXCL12 and LPS-matured DCs towards CCL21 was reduced in sgk3^-/- as compared to sgk3^+/+ DCs. Migration of sgk3^+/+ DCs was further sensitive to SOC channel inhibitor 2-APB (50 µM) and to STIM1/STIM2 knock-down. Conclusion: SGK3 contributes to the regulation of store-operated Ca²⁺ entry into and migration of dendritic cells, effects at least partially mediated through SGK3-dependent upregulation of STIM2 expression.

SGK1 Sensitivity of Platelet Migration

Recent observations pointed to the ability of platelets to migrate and thus to invade the inflamed vascular wall. Platelet migration could be stimulated by stromal cell-derived factor-1 (SDF-1), an effect dependent on phosphatidylinositide-3-kinase (PI3K) and paralleled by activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP). Migration is inhibited by vinculin, which is similarly regulated by phosphorylation. PI3K-sensitive kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored whether SGK1 modifies WASP and vinculin phosphorylation in murine platelets and participates in the regulation of platelet migration. Platelets were isolated from gene-targeted mice lacking SGK1 (sgk1-/-) and from their wild type littermates (sgk1+/+). Platelet migration stimulated with SDF-1 was significantly less pronounced in sgk1-/-platelets than in sgk1+/+ platelets. Moreover, SDF-1 significantly induced WASP phosphorylation, an effect again reduced in platelets lacking SGK1. Phosphorylation of vinculin was significantly enhanced in sgk1-/-platelets and was significantly reduced following treatment of platelets with Ca2+ chelator BAPTA. Immunohistochemical analysis of in vivo experiments in intestinal vessels after vascular inflammation revealed that transmigration of platelets into inflamed vessel walls was significantly less pronounced in sgk1-/-than in sgk1+/+ mice. In conclusion, SGK1 is a powerful regulator of platelet migration.

NH4Cl Treatment Prevents Tissue Calcification in Klotho Deficiency

Klotho, a cofactor in suppressing 1,25(OH)2D3 formation, is a powerful regulator of mineral metabolism. Klotho-hypomorphic mice (kl/kl) exhibit excessive plasma 1,25(OH)2D3, Ca(2+), and phosphate concentrations, severe tissue calcification, volume depletion with hyperaldosteronism, and early death. Calcification is paralleled by overexpression of osteoinductive transcription factor Runx2/Cbfa1, Alpl, and senescence-associated molecules Tgfb1, Pai-1, p21, and Glb1. Here, we show that NH4Cl treatment in drinking water (0.28 M) prevented soft tissue and vascular calcification and increased the life span of kl/kl mice >12-fold in males and >4-fold in females without significantly affecting extracellular pH or plasma concentrations of 1,25(OH)2D3, Ca(2+), and phosphate. NH4Cl treatment significantly decreased plasma aldosterone and antidiuretic hormone concentrations and reversed the increase of Runx2/Cbfa1, Alpl, Tgfb1, Pai-1, p21, and Glb1 expression in aorta of kl/kl mice. Similarly, in primary human aortic smooth muscle cells (HAoSMCs), NH4Cl treatment reduced phosphate-induced mRNA expression of RUNX2/CBFA1, ALPL, and senescence-associated molecules. In both kl/kl mice and phosphate-treated HAoSMCs, levels of osmosensitive transcription factor NFAT5 and NFAT5-downstream mediator SOX9 were higher than in controls and decreased after NH4Cl treatment. Overexpression of NFAT5 in HAoSMCs mimicked the effect of phosphate and abrogated the effect of NH4Cl on SOX9, RUNX2/CBFA1, and ALPL mRNA expression. TGFB1 treatment of HAoSMCs upregulated NFAT5 expression and prevented the decrease of phosphate-induced NFAT5 expression after NH4Cl treatment. In conclusion, NH4Cl treatment prevents tissue calcification, reduces vascular senescence, and extends survival of klotho-hypomorphic mice. The effects of NH4Cl on vascular osteoinduction involve decrease of TGFB1 and inhibition of NFAT5-dependent osteochondrogenic signaling.

Impact of Bicarbonate, Ammonium Chloride, and Acetazolamide on Hepatic and Renal SLC26A4 Expression

SLC26A4 encodes pendrin, a transporter exchanging anions such as chloride, bicarbonate, and iodide. Loss of function mutations of SLC26A4 cause Pendred syndrome characterized by hearing loss and enlarged vestibular aqueducts as well as variable hypothyroidism and goiter. In the kidney, pendrin is expressed in the distal nephron and accomplishes HCO3- secretion and Cl- reabsorption. Renal pendrin expression is regulated by acid-base balance. The liver contributes to acid-base regulation by producing or consuming glutamine, which is utilized by the kidney for generation and excretion of NH4+, paralleled by HCO3- formation. Little is known about the regulation of pendrin in liver. The present study thus examined the expression of Slc26a4 in liver and kidney of mice drinking tap water without or with NaHCO3 (150 mM), NH4Cl (280 mM) or acetazolamide (3.6 mM) for seven days. As compared to Gapdh transcript levels, Slc26a4 transcript levels were moderately lower in liver than in renal tissue. Slc26a4 transcript levels were not significantly affected by NaHCO3 in liver, but significantly increased by NaHCO3 in kidney. Pendrin protein expression was significantly enhanced in kidney and reduced in liver by NaHCO3. Slc26a4 transcript levels were significantly increased by NH4Cl and acetazolamide in liver, and significantly decreased by NH4Cl and by acetazolamide in kidney. NH4Cl and acetazolamide reduced pendrin protein expression significantly in kidney, but did not significantly modify pendrin protein expression in liver. The observations point to expression of pendrin in the liver and to opposite effects of acidosis on pendrin transcription in liver and kidney.

Acid Sphingomyelinase Regulates Platelet Cell Membrane Scrambling, Secretion, and Thrombus Formation

Objective— Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation. Approach and Results— Collagen-related peptide–induced or thrombin-induced ATP release and P-selectin exposure were significantly blunted in platelets from Asm-deficient mice (Smpd1 −/−) when compared with platelets from wild-type mice (Smpd1 +/+ ). Moreover, phosphatidylserine exposure and thrombin generation were significantly less pronounced in Smpd1 −/− platelets than in Smpd1 +/+ platelets. In contrast, platelet integrin &agr;IIb&bgr;3 activation and aggregation, as well as activation-dependent Ca2+ flux, were not significantly different between Smpd1 −/− and Smpd1 +/+ platelets. In vitro thrombus formation at shear rates of 1700 s−1 and in vivo thrombus formation after FeCl3 injury were significantly blunted in Smpd1 −/− mice while bleeding time was unaffected. Asm-deficient platelets showed significantly reduced activation-dependent ceramide formation, whereas exogenous ceramide rescued diminished platelet secretion and thrombus formation caused by Asm deficiency. Treatment of Smpd1 +/+ platelets with bacterial sphingomyelinase (0.01 U/mL) increased, whereas treatment with functional acid sphingomyelinase-inhibitors, amitriptyline or fluoxetine (5 &mgr;mol/L), blunted activation-dependent platelet degranulation, phosphatidylserine exposure, and thrombus formation. Impaired degranulation and thrombus formation of Smpd1 −/− platelets were again overcome by exogenous bacterial sphingomyelinase. Conclusions— Acid sphingomyelinase is a completely novel element in the regulation of platelet plasma membrane properties, secretion, and thrombus formation.

Akt2 Deficiency is Associated with Anxiety and Depressive Behavior in Mice

Background: The economic burden associated with major depressive disorder and anxiety disorders render both disorders the most common and debilitating psychiatric illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology, successful treatment and prevention of these highly associated disorders have not been identified. Akt2 is a key protein in the phosphatidylinositide-3 (PI3K) / glycogen synthase 3 kinase (GSK3) signaling pathway, which in turn is involved in brain-derived neurotrophic factor (BDNF) effects on fear memory, mood stabilisation and action of several antidepressant drugs. The present study thus explored the impact of Akt2 on behaviour of mice. Methods: Behavioural studies (Open-Field, Light-Dark box, O-Maze, Forced Swimming Test, Emergence Test, Object Exploration Test, Morris Water Maze, Radial Maze) have been performed with Akt2 knockout mice (akt-/-) and corresponding wild type mice (akt+/+). Results: Anxiety and depressive behavior was significantly higher in akt-/- than in akt+/+ mice. The akt-/- mice were cognitively unimpaired but displayed increased anxiety in several behavioral tests (O-Maze test, Light-Dark box, Open Field test). Moreover, akt-/- mice spent more time floating in the Forced Swimming test, which is a classical feature of experimental depression. Conclusion: Akt2 might be a key factor in the pathophysiology of depression and anxiety.

1,25( OH)(2) vitamin D-3-dependent inhibition of platelet Ca2+ signaling and thrombus formation in klotho-deficient mice

Platelets are activated by increased cytosolic Ca2+ concentration ([Ca2+]i) following store‐operated calcium entry (SOCE) accomplished by calcium‐release‐activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca2+ transport is regulated by 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 formation is inhibited by klotho and excessive in klotho‐deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca2+ signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl‐mice were analyzed by RT‐PCR, Western blotting, confocal microscopy, Fura‐2‐fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist‐induced [Ca2+]i increase, activation‐dependent degranulation, integrin αIIbβ3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27–90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38–73%) and 1,25(OH)2D3‐treated WT megakaryocytes. Nuclear NF‐κB subunit p50/p65 abundance was significantly reduced in kl/kl‐megakaryocytes (by 51–76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG‐01 cells (by 46–97%). Low‐vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca2+ signaling and activation, an effect at least partially due to 1,25(OH)2D3‐dependent down‐regulation of NF‐κB activity and STIM1/Orai1 expression in megakaryocytes.—Borst, O., Münzer, P., Schmid, E., Schmidt, E.‐M., Russo, A., Walker, B., Yang, W., Leibrock, C., Szteyn, K., Schmidt, S., Elvers, M., Faggio, C., Shumilina, E., Kuro‐o, M., Gawaz, M., Lang, F. 1,25(OH)2 vitamin D3‐dependent inhibition of platelet Ca2+ signaling and thrombus formation in klotho‐deficient mice. FASEB J. 28, 2108–2119 (2014). www.fasebj.org