Extracellular sodium regulates fibroblast growth factor 23 (FGF23) formation.

The bone-derived hormone fibroblast growth factor-23 (FGF23) has recently received much attention due to its association with chronic kidney disease and cardiovascular disease progression. Extracellular sodium concentration ([Na+]) plays a significant role in bone metabolism. Hyponatremia (lower serum [Na+]) has recently been shown to be independently associated with FGF23 levels in patients with chronic systolic heart failure. However, nothing is known about the direct impact of [Na+] on FGF23 production. Here, we show that an elevated [Na+] (+20 mM) suppressed FGF23 formation, whereas low [Na+] (-20 mM) increased FGF23 synthesis in the osteoblast-like cell lines UMR-106 and MC3T3-E1. Similar bidirectional changes in FGF23 abundance were observed when osmolality was altered by mannitol but not by urea, suggesting a role of tonicity in FGF23 formation. Moreover, these changes in FGF23 were inversely proportional to the expression of NFAT5 (nuclear factor of activated T cells-5), a transcription factor responsible for tonicity-mediated cellular adaptations. Furthermore, arginine vasopressin, which is often responsible for hyponatremia, did not affect FGF23 production. Next, we performed a comprehensive and unbiased RNA-seq analysis of UMR-106 cells exposed to low versus high [Na+], which revealed several novel genes involved in cellular adaptation to altered tonicity. Additional analysis of cells with Crisp-Cas9-mediated NFAT5 deletion indicated that NFAT5 controls numerous genes associated with FGF23 synthesis, thereby confirming its role in [Na+]-mediated FGF23 regulation. In line with these in vitro observations, we found that hyponatremia patients have higher FGF23 levels. Our results suggest that [Na+] is a critical regulator of FGF23 synthesis.

Structural mechanism of cGAS inhibition by the nucleosome.

The DNA sensor cyclic GMP-AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer1. Upon activation by double-stranded DNA, cytosolic cGAS produces 2'3' cGMP-AMP, which triggers the induction of inflammatory cytokines and type I interferons 2-7. cGAS is also present inside the cell nucleus, which is replete with genomic DNA8, where chromatin has been implicated in restricting its enzymatic activity9. However, the structural basis for inhibition of cGAS by chromatin remains unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes. cGAS makes extensive contacts with both the acidic patch of the histone H2A-H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also find cGAS engaged to a second nucleosome in trans. Mechanistically, binding of the nucleosome locks cGAS into a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS-acidic patch interface are sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash the activity of cGAS on genomic DNA in living cells. Our work uncovers the structural basis of the interaction between cGAS and chromatin and details a mechanism that permits self-non-self discrimination of genomic DNA by cGAS.

Tanshinone IIA stimulates erythrocyte phosphatidylserine exposure.

Tanshinone IIA, an antimicrobial, antioxidant, antianaphylactic, antifibrotic, vasodilating, antiatherosclerotic, organo-protective and antineoplastic component from the rhizome of Salvia miltiorrhiza, is known to trigger apoptosis of tumor cells. Tanshinone IIA is effective in part through mitochondrial depolarization and altered gene expression. Erythrocytes lack mitochondria and nuclei but may undergo eryptosis, an apoptosis-like suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity, ATP depletion and ceramide formation. The present study explored, whether tanshinone IIA elicits eryptosis. Cytosolic Ca(2+)-concentration was determined from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from binding of fluorescent annexin V, hemolysis from hemoglobin concentration in the supernatant, ATP concentration utilizing luciferin-luciferase and ceramide formation utilizing fluorescent anticeramide antibodies. Clearance of circulating erythrocytes was estimated by CFSE-labeling. A 48 h exposure to tanshinone IIA (≥10 µM) significantly increased cytosolic Ca(2+)-concentration, decreased ATP concentration (25 µM), increased lactate concentration (25 µM), increased ceramide formation (25 µM), decreased forward scatter, increased annexin-V-binding and increased (albeit to a much smaller extent) hemolysis. The effect of 25 µM tanshinone IIA on annexin-V binding was partially reversed in the nominal absence of Ca(2+). Labelled tanshinone IIA-treated erythrocytes were more rapidly cleared from the circulating blood in comparison to untreated erythrocytes. The present observations reveal a completely novel effect of tanshinone IIA, i.e. triggering of Ca(2+) entry, ATP depletion and ceramide formation in erythrocytes, events eventually leading to eryptosis with cell shrinkage and cell membrane scrambling.

Glucocorticoids enhance intestinal glucose uptake via the dimerized glucocorticoid receptor in enterocytes.

Glucocorticoid (GC) treatment of inflammatory disorders, such as inflammatory bowel disease, causes deranged metabolism, in part by enhanced intestinal resorption of glucose. However, the underlying molecular mechanism is poorly understood. Hence, we investigated transcriptional control of genes reported to be involved in glucose uptake in the small intestine after GC treatment and determined effects of GC on electrogenic glucose transport from transepithelial currents. GR(villinCre) mice lacking the GC receptor (GR) in enterocytes served to identify the target cell of GC treatment and the requirement of the GR itself; GR(dim) mice impaired in dimerization and DNA binding of the GR were used to determine the underlying molecular mechanism. Our findings revealed that oral administration of dexamethasone to wild-type mice for 3 d increased mRNA expression of serum- and GC-inducible kinase 1, sodium-coupled glucose transporter 1, and Na(+)/H(+) exchanger 3, as well as electrogenic glucose transport in the small intestine. In contrast, GR(villinCre) mice did not respond to GC treatment, neither with regard to gene activation nor to glucose transport. GR(dim) mice were also refractory to GC, because dexamethasone treatment failed to increase both, gene expression and electrogenic glucose transport. In addition, the rise in blood glucose levels normally observed after GC administration was attenuated in both mutant mouse strains. We conclude that enhanced glucose transport in vivo primarily depends on gene regulation by the dimerized GR in enterocytes, and that this mechanism contributes to GC-induced hyperglycemia.

SGK1-dependent stimulation of intestinal SGLT1 activity by vitamin D.

The serum- and glucocorticoid-inducible kinase SGK1 has previously been shown to mediate the glucocorticoid-dependent stimulation of several intestinal transport systems including the electrogenic glucose transporter SGLT1. In squamous carcinoma cells, SGK1 expression is stimulated by 1,25(OH)2D3, the biologically active metabolite of vitamin D. The present study explored whether vitamin D influences the intestinal SGLT1 activity. Jejunal SGLT1 activity was determined by Ussing chamber experiments. Under a normal diet, the electrogenic glucose transport was similar in SGK1 knockout (sgk1 ( -/- )) and wild type mice (sgk1 ( +/+ )). Following a vitamin D-rich diet (14 days 10,000 I.U. vitamin D), the SGK1 transcript levels as well as the SGLT1 protein abundance were increased in sgk1(+/+) mice. Moreover, SGLT1 activity was increased in sgk1(+/+) mice but not in sgk1(-/-) mice following a vitamin D-rich diet. Furthermore, an oral glucose load was followed by an increase in the plasma glucose concentration to significantly higher values in sgk1(+/+) mice treated with a vitamin D-rich diet than in untreated sgk1(+/+) mice. In conclusion, vitamin D treatment upregulates the expression of SGK1, which in turn enhances SGLT1 activity.

PKB/SGK-resistant GSK3 enhances phosphaturia and calciuria.

Insulin and IGF1-dependent signaling activates protein kinase B and serum and glucocorticoid inducible kinase (PKB/SGK), which together phosphorylate and inactivate glycogen synthase kinase GSK3. Because insulin and IGF1 increase renal tubular calcium and phosphorus reabsorption, we examined GSK3 regulation of phosphate transporter activity and determined whether PKB/SGK inactivates GSK3 to enhance renal phosphate and calcium transport. Overexpression of GSK3 and the phosphate transporter NaPi-IIa in Xenopus oocytes decreased electrogenic phosphate transport compared with NaPi-IIa-expressing oocytes. PKB/SGK serine phosphorylation sites in GSK3 were mutated to alanine to create gsk3(KI) mice resistant to PKB/SGK inactivation. Compared with wildtype animals, gsk3(KI) animals exhibited greater urinary phosphate and calcium clearances with higher excretion rates and lower plasma concentrations. Isolated brush border membranes from gsk3(KI) mice showed less sodium-dependent phosphate transport and Na-phosphate co-transporter expression. Parathyroid hormone, 1,25-OH vitamin D levels, and bone mineral density were decreased in gsk3(KI) mice, suggesting a global dysregulation of bone mineral metabolism. Taken together, PKB/SGK phosphorylation of GSK3 increases phosphate transporter activity and reduces renal calcium and phosphate loss.