IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway.
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ABSTRACT: Fluid and HCO(3)(-) secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO(3)(-) in secretory glands is fueled by Na(+)/HCO(3)(-) cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl(-)/HCO(3)(-) exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO(3)(-) secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase-regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.
Project description:Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently identified with-no-lysine kinase (WNK)-oxidative stress-responsive kinase 1 (OSR1)/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter (NCC) phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the phosphatidylinositol 3-kinase/Akt signaling cascade in the kidney in response to hyperinsulinemia. A phosphatidylinositol 3-kinase inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific phosphatidylinositol 3-kinase inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions, such as the metabolic syndrome.
Project description:Fluid and HCO3(-) secretion is a vital function of secretory epithelia, involving basolateral HCO3(-) entry through the Na(+)-HCO3(-) cotransporter (NBC) NBCe1-B, and luminal HCO3(-) exit mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) Cl(-)/HCO3(-) exchangers. HCO3(-) secretion is highly regulated, with the WNK/SPAK kinase pathway setting the resting state and the IRBIT/PP1 pathway setting the stimulated state. However, we know little about the relationships between the WNK/SPAK and IRBIT/PP1 sites in the regulation of the transporters. The first 85 N-terminal amino acids of NBCe1-B function as an autoinhibitory domain. Here we have identified a positively charged module within NBCe1-B(37-65) that is conserved in NBCn1-A and all 20 members of the NBC superfamily except NBCe1-A. This module is required for the interaction and activation of NBCe1-B and NBCn1-A by IRBIT and their regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Activation of the transporters by IRBIT and PIP2 is nonadditive but complementary. Phosphorylation of Ser65 mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 is required for regulation by IRBIT and SPAK. Sequence searches using the NBCe1-B regulatory module as a template identified a homologous sequence in the CFTR R domain and Slc26a6 sulfat transporter and antisigma factor antagonist (STAS) domain. Accordingly, the R and STAS domains bind IRBIT, and the R domain is required for activation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in a conserved domain of the NBC that may be present in other HCO3(-) transporters and thus in the regulation of epithelial fluid and HCO3(-) secretion.
Project description:Mutations within the with-no-K(Lys) (WNK) kinases cause Gordon's syndrome characterized by hypertension and hyperkalaemia. WNK kinases phosphorylate and activate the STE20/SPS1-related proline/alanine-rich kinase (SPAK) protein kinase, which phosphorylates and stimulates the key Na(+):Cl(-) cotransporter (NCC) and Na(+):K(+):2Cl(-) cotransporters (NKCC2) cotransporters that control salt reabsorption in the kidney. To define the importance of this pathway in regulating blood pressure, we generated knock-in mice in which SPAK cannot be activated by WNKs. The SPAK knock-in animals are viable, but display significantly reduced blood pressure that was salt-dependent. These animals also have markedly reduced phosphorylation of NCC and NKCC2 cotransporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in messenger RNA (mRNA) levels. On a normal Na(+)-diet, the SPAK knock-in mice were normokalaemic, but developed mild hypokalaemia when the renin-angiotensin system was activated by a low Na(+)-diet. These observations establish that SPAK plays an important role in controlling blood pressure in mammals. Our results imply that SPAK inhibitors would be effective at reducing blood pressure by lowering phosphorylation as well as expression of NCC and NKCC2. See accompanying Closeup by Maria Castañeda-Bueno and Gerald Gamba (DOI 10.1002/emmm.200900059).
Project description:The role of Cl- as an intracellular signaling ion has been increasingly recognized in recent years. One of the currently best described roles of Cl- in signaling is the modulation of the With-No-Lysine (K) (WNK) - STE20-Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1) - Cation-Coupled Cl- Cotransporters (CCCs) cascade. Binding of a Cl- anion to the active site of WNK kinases directly modulates their activity, promoting their inhibition. WNK activation due to Cl- release from the binding site leads to phosphorylation and activation of SPAK/OSR1, which in turn phosphorylate the CCCs. Phosphorylation by WNKs-SPAK/OSR1 of the Na+-driven CCCs (mediating ions influx) promote their activation, whereas that of the K+-driven CCCs (mediating ions efflux) promote their inhibition. This results in net Cl- influx and feedback inhibition of WNK kinases. A wide variety of alterations to this pathway have been recognized as the cause of several human diseases, with manifestations in different systems. The understanding of WNK kinases as Cl- sensitive proteins has allowed us to better understand the mechanistic details of regulatory processes involved in diverse physiological phenomena that are reviewed here. These include cell volume regulation, potassium sensing and intracellular signaling in the renal distal convoluted tubule, and regulation of the neuronal response to the neurotransmitter GABA.
Project description:Chronic high blood pressure (hypertension) is the most common disease in the Unites States. While several classes of drugs exist to treat it, many patients (up to 10 million Americans) respond poorly to therapy, even when multiple classes are used. Recent evidence suggests that a significant portion of patients will always remain hypertensive despite maximum therapy with the drugs currently available. Therefore, there is a pressing need to develop novel antihypertensive agents. One limitation has been the identification of new targets, a limitation that has been overcome by recent insights into the mechanisms underlying monogenic forms of hypertension. The disease familial hyperkalemic hypertension is caused by mutations in with-no-lysine (WNK) kinases 1 and 4 and in cullin-3 and kelch-like 3, components of an E3 ubiquitin ligase complex that promotes WNK kinase degradation. The study of the mechanisms by which this pathway regulates blood pressure has identified several candidates for the development of new antihypertensive agents. This pathway is particularly attractive since its inhibition may not only reduce renal sodium reabsorption along multiple segments but may also reduce vascular tone. Here, we will describe the mechanisms by which this pathway regulate blood pressure and discuss the potential of targeting it to develop new antihypertensive drugs.
Project description:Upon activation by with-no-lysine kinases, STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na(+)-Cl(-) cotransporter (NCC) and Na(+)-K(+)-2Cl(-) cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.
Project description:The renal thiazide-sensitive NaCl cotransporter (NCC) is the major salt transport pathway in the distal convoluted tubule of the mammalian nephron. NCC activity is critical for modulation of arterial blood pressure and serum potassium levels. Reduced activity of NCC in genetic diseases results in arterial hypotension and hypokalemia, while increased activity results in genetic diseases featuring hypertension and hyperkalemia. Several hormones and physiological conditions modulate NCC activity through a final intracellular complex pathway involving kinases and ubiquitin ligases. A substantial amount of work has been conducted to understand this pathway in the last 15 yr, but advances over the last 3 yr have helped to begin to understand how these regulatory proteins interact with each other and modulate the activity of this important cotransporter. In this review, we present the current model of NCC regulation by the Cullin 3 protein/Kelch-like 3 protein/with no lysine kinase/STE20-serine-proline alanine-rich kinase (CUL3/KELCH3-WNK-SPAK) pathway. We present a review of all genetically altered mice that have been used to translate most of the proposals made from in vitro experiments into in vivo observations that have helped to elucidate the model at the physiological level. Many questions have been resolved, but some others will require further models to be constructed. In addition, unexpected observations in mice have raised new questions and identified regulatory pathways that were previously unknown.
Project description:Mammalian Ste20-like proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinases phosphorylate and regulate cation-coupled Cl(-) cotransporter activity in response to cell volume changes. SPAK and OSR1 are activated via phosphorylation by upstream with-no-lysine (WNK) kinases. In Caenorhabditis elegans, the SPAK/OSR1 ortholog germinal center kinase (GCK)-3 binds to and regulates the activity of the cell volume- and meiotic cell cycle-dependent ClC anion channel CLH-3b. We tested the hypothesis that WNK kinases function in the GCK-3/CLH-3b signaling cascade. CLH-3b heterologously expressed in human embryonic kidney (HEK) cells was unaffected by coexpression with the single C. elegans WNK kinase, WNK-1, or kinase-dead WNK-1 dominant-negative mutants. RNA interference (RNAi) knockdown of the single Drosophila WNK kinase had no effect on the activity of CLH-3b expressed in Drosophila S2 cells. Similarly, RNAi silencing of C. elegans WNK-1 had no effect on basal or cell volume-sensitive activity of CLH-3b expressed endogenously in worm oocytes. Previous yeast 2-hybrid studies suggested that ERK kinases may function upstream of GCK-3. Pharmacological inhibition of ERK signaling disrupted CLH-3b activity in HEK cells in a GCK-3-dependent manner. RNAi silencing of the C. elegans ERK kinase MPK-1 or the ERK phosphorylating/activating kinase MEK-2 constitutively activated native CLH-3b. MEK-2 and MPK-1 play important roles in regulating the meiotic cell cycle in C. elegans oocytes. Cell cycle-dependent changes in MPK-1 correlate with the pattern of CLH-3b activation observed during oocyte meiotic maturation. We postulate that MEK-2/MPK-1 functions upstream from GCK-3 to regulate its activity during cell volume and meiotic cell cycle changes.
Project description:BackgroundWorsened stroke outcomes with hypertension comorbidity are insensitive to blood pressure-lowering therapies. In an experimental stroke model with comorbid hypertension, we investigated causal roles of ang II (angiotensin II)-mediated stimulation of the brain WNK (with no lysine [K] kinases)-SPAK (STE20/SPS1-related proline/alanine-rich kinase)-NKCC1 (Na-K-Cl cotransporter) complex in worsened outcomes.MethodsSaline- or ang II-infused C57BL/6J male mice underwent stroke induced by permanent occlusion of the distal branches of the middle cerebral artery. Mice were randomly assigned to receive either vehicle dimethyl sulfoxide/PBS (2 mL/kg body weight/day, IP), a novel SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (ZT-1a' 5 mg/kg per day, IP) or a NF-κB (nuclear factor-κB) inhibitor TAT-NBD (transactivator of transcription-NEMO-binding domain' 20 mg/kg per day, IP). Activation of brain NF-κB and WNK-SPAK-NKCC1 cascade as well as ischemic stroke outcomes were examined.ResultsStroke triggered a 2- to 5-fold increase of WNK (isoforms 1, 2, 4), SPAK/OSR1 (oxidative stress-responsive kinase 1), and NKCC1 protein in the ang II-infused hypertensive mouse brains at 24 hours after stroke, which was associated with increased nuclear translocation of phospho-NF-κB protein in the cortical neurons (a Pearson correlation r of 0.77, P<0.005). The upregulation of WNK-SPAK-NKCC1 cascade proteins resulted from increased NF-κB recruitment on Wnk1, Wnk2, Wnk4, Spak, and Nkcc1 gene promoters and was attenuated by NF-κB inhibitor TAT-NBD. Poststroke administration of SPAK inhibitor ZT-1a significantly reduced WNK-SPAK-NKCC1 complex activation, brain lesion size, and neurological function deficits in the ang II-hypertensive mice without affecting blood pressure and cerebral blood flow.ConclusionsThe ang II-induced stimulation of NF-κB transcriptional activity upregulates brain WNK-SPAK-NKCC1 cascade and contributes to worsened ischemic stroke outcomes, illustrating the brain WNK-SPAK-NKCC1 complex as a therapeutic target for stroke with comorbid hypertension.
Project description:INTRODUCTION:The mammalian SPS1-related proline/alanine-rich serine-threonine kinase SPAK (STK39) modulates ion transport across and between epithelial cells in response to environmental stimuli such osmotic stress and inflammation. Research over the last decade has established a central role for SPAK in the regulation of ion and water transport in the distal nephron, colonic crypts, and pancreatic ducts, and has implicated deregulated SPAK signaling in NaCl-sensitive hypertension, ulcerative colitis and Crohn's disease, and cystic fibrosis. Areas covered: We review recent advances in our understanding of the role of SPAK kinase in the regulation of epithelial transport. We highlight how SPAK signaling - including its upstream Cl- sensitive activators, the WNK kinases, and its downstream ion transport targets, the cation- Cl- cotransporters contribute to human disease. We discuss prospects for the pharmacotherapeutic targeting of SPAK kinase in specific human disorders that feature impaired epithelial homeostasis. Expert opinion: The development of novel drugs that antagonize the SPAK-WNK interaction, inhibit SPAK kinase activity, or disrupt SPAK kinase activation by interfering with its binding to MO25?/? could be useful adjuncts in essential hypertension, inflammatory colitis, and cystic fibrosis.