Glyburide inhibits the Cryopyrin/Nalp3 inflammasome

Glyburide, a sulfonylurea drug commonly used to treat type 2 diabetes, shuts down IL-1β secretion by preventing Cyropyrin activation

Acute Insulin Stimulation Induces Phosphorylation of the Na-Cl Cotransporter in Cultured Distal mpkDCT Cells and Mouse Kidney

The NaCl cotransporter (NCC) is essential for sodium reabsorption at the distal convoluted tubules (DCT), and its phosphorylation increases its transport activity and apical membrane localization. Although insulin has been reported to increase sodium reabsorption in the kidney, the linkage between insulin and NCC phosphorylation has not yet been investigated. This study examined whether insulin regulates NCC phosphorylation. In cultured mpkDCT cells, insulin increased phosphorylation of STE20/SPS1-related proline-alanine-rich kinase (SPAK) and NCC in a dose-dependent manner. This insulin-induced phosphorylation of NCC was suppressed in WNK4 and SPAK knockdown cells. In addition, Ly294002, a PI3K inhibitor, decreased the insulin effect on SPAK and NCC phosphorylation, indicating that insulin induces phosphorylation of SPAK and NCC through PI3K and WNK4 in mpkDCT cells. Moreover, acute insulin administration to mice increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), SPAK and NCC in the kidney. Time-course experiments in mpkDCT cells and mice suggested that SPAK is upstream of NCC in this insulin-induced NCC phosphorylation mechanism, which was confirmed by the lack of insulin-induced NCC phosphorylation in SPAK knockout mice. Moreover, insulin administration to WNK4 hypomorphic mice did not increase phosphorylation of OSR1, SPAK and NCC in the kidney, suggesting that WNK4 is also involved in the insulin-induced OSR1, SPAK and NCC phosphorylation mechanism in vivo. The present results demonstrated that insulin is a potent regulator of NCC phosphorylation in the kidney, and that WNK4 and SPAK are involved in this mechanism of NCC phosphorylation by insulin

Widespread modulation of gene expression by copy number variation in skeletal muscle

Copy number variation (CNV) is a frequently observed deviation from the diploid state due to duplication or deletion of genomic regions. Although intensively analyzed for association with diseases and production traits, the specific mechanisms and extent by which such variations affect the phenotype are incompletely understood. We present an integrative study on CNV and genome-wide gene expression in Brazilian Bos indicus cattle. We analyzed CNVs inferred from SNP-chip data for effects on gene expression measured with RNA-seq in skeletal muscle samples of 183 steers. Local effects, where expression changes coincided with CNVs in the respective genes, were restricted to immune genes. Distal effects were attributable to several high-impact CNVs that modulated remote expression in an orchestrated and intertwined fashion. These CNVs were located in the vicinity of major skeletal muscle pathway regulators and associated genes were enriched for proteolysis, autophagy, and muscle structure development. From association analysis between CNVs and several meat quality and production traits, we found CNV-associated expression effects to also manifest at the phenotype level. Based on genome sequences of the population founders, we further demonstrate that CNVs with impact on expression and phenotype are passed on from one generation to another

Phosphorylation and Transport in the Na-K-2Cl Cotransporters, NKCC1 and NKCC2A, Compared in HEK-293 Cells

Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K+ (86Rb+) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37°C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl−] media to reduce cell [Cl−] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na+-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates

Functional kinomics establishes a critical node of volume-sensitive cation-Cl^- cotransporter regulation in the mammalian brain

This is the final version of the article. Available from the publisher via the DOI in this record.There is another record in ORE for this publication: http://hdl.handle.net/10871/33424Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation – a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl−-sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl− uptake and stimulation of KCC3-mediated Cl− extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought “Cl−/volume-sensitive kinase” of the cation-Cl− cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.We thank the excellent technical support of the MRC-Protein Phosphorylation and Ubiquitylation Unit (PPU) DNA Sequencing Service (coordinated by Nicholas Helps), the MRC-PPU tissue culture team (coordinated by Laura Fin), the Division of Signal Transduction Therapy (DSTT) antibody purification teams (coordinated by Hilary McLauchlan and James Hastie). We are grateful to the MRC PPU Proteomics facility (coordinated by David Campbell, Robert Gourlay and Joby Varghese). We thank for support the Medical Research Council (MC_UU_12016/2; DRA) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer; DRA). We thank Thomas J. Jentsch (Max-Delbrück-Centrum für Molekulare Medizin) for providing the KCC1/3 double KO mice and his reading of this manuscript. We thank Nathaniel Grey (Harvard) for providing the kinase inhibitor library used in this study (NIH LINCS Program grant U54HL127365). This work was also supported by a Harvard-MIT Neuroscience Grant (to KTK/SJE)

Inflammation-dependent cerebrospinal fluid hypersecretion by the choroid plexus epithelium in posthemorrhagic hydrocephalus

This is the author accepted manuscript. The final version is available from Springer Nature via the DOI in this recordThere is another record in ORE for this publication: http://hdl.handle.net/10871/33419The choroid plexus epithelium (CPE) secretes higher volumes of fluid (cerebrospinal fluid, CSF) than any other epithelium and simultaneously functions as the blood-CSF barrier to gate immune cell entry into the central nervous system. Posthemorrhagic hydrocephalus (PHH), an expansion of the cerebral ventricles due to CSF accumulation following intraventricular hemorrhage (IVH), is a common disease usually treated by suboptimal CSF shunting techniques. PHH is classically attributed to primary impairments in CSF reabsorption, but little experimental evidence supports this concept. In contrast, the potential contribution of CSF secretion to PHH has received little attention. In a rat model of PHH, we demonstrate that IVH causes a Toll-like receptor 4 (TLR4)- and NF-κB-dependent inflammatory response in the CPE that is associated with a ∼3-fold increase in bumetanide-sensitive CSF secretion. IVH-induced hypersecretion of CSF is mediated by TLR4-dependent activation of the Ste20-type stress kinase SPAK, which binds, phosphorylates, and stimulates the NKCC1 co-transporter at the CPE apical membrane. Genetic depletion of TLR4 or SPAK normalizes hyperactive CSF secretion rates and reduces PHH symptoms, as does treatment with drugs that antagonize TLR4-NF-κB signaling or the SPAK-NKCC1 co-transporter complex. These data uncover a previously unrecognized contribution of CSF hypersecretion to the pathogenesis of PHH, demonstrate a new role for TLRs in regulation of the internal brain milieu, and identify a kinase-regulated mechanism of CSF secretion that could be targeted by repurposed US Food and Drug Administration (FDA)-approved drugs to treat hydrocephalus.We thank D.R. Alessi (Dundee) and R.P. Lifton (Rockefeller) for their support. K.T.K. is supported by the March of Dimes Basil O'Connor Award, a Simons Foundation SFARI Grant, the Hydrocephalus Association Innovator Award, and the NIH (4K12NS080223-05). J.M.S. is supported by the National Institute of Neurological Disorders and Stroke (NINDS) (NS060801; NS061808) and the US Department of Veterans Affairs (1BX002889); R.M. is supported by the Howard Hughes Medical Institute