With-no-lysine Kinase 1 Research Articles

Bilobalide Activates Autophagy and Enhances the Efficacy of Bone Marrow Mesenchymal Stem Cells on Spinal Cord Injury Via Upregulating FMRP to Promote WNK1 mRNA Decay.

Transplantation of bone marrow mesenchymal stem cells (BMSCs) represents an encouraging strategy for the repair of spinal cord injury (SCI), however, its effectiveness on treating SCI remains controversial. Bilobalide isolated from Ginkgo biloba leaves shows significant neuroprotective effects. We examined the role and underlying mechanism of bilobalide in the efficacy of BMSC transplantation on SCI. Primary BMSCs were isolated from neonatal rats, and cell viability was assessed by MTT assay. Neuronal markers (MAP-2, NeuN, NSE and Tuj1), autophagy markers (LC3 and Beclin1), and Fragile X mental retardation protein (FMRP)/With-no-lysine kinase-1 (WNK1) signaling were measured using RT-qPCR and western blotting. The relationship of FMRP and WNK1 was estimated by RNA immunoprecipitation, while WNK1 mRNA stability was assessed with actinomycin D assay. In a SCI rat model, tissue injury was examined using HE and Nissl staining. Bilobalide treatment facilitated neural differentiation of BMSCs, as well as enhanced autophagy and inhibited WNK1 signaling. The promotive effect of bilobalide on BMSC differentiation was antagonized when overexpressing WNK1 or inhibiting autophagy. Bilobalide upregulated FMRP to promote WNK1 mRNA decay, thus reducing WNK1 expression. FMRP knockdown reversed the promoted functions of bilobalide on autophagy and neuronal differentiation in BMSCs. Additionally, compared to either monotherapy, simultaneous treatments with bilobalide and BMSCs further facilitated autophagy and neuronal differentiation, thereby enhancing the repair of SCI in rats. Bilobalide enhances autophagy activity to promote BMSC neuronal differentiation via FMRP/WNK1 axis, thus improving functional recovery following SCI, which indicates a promising therapeutic approach for SCI.

The biology of water homeostasis.

Water homeostasis is controlled by a brain-kidney axis that consists of central osmoreceptors, synthesis and secretion of arginine vasopressin (AVP) and AVP-responsive aquaporin-2 (AQP2) water channels in kidney collecting duct principal cells that facilitate water reabsorption. In addition to AVP, thirst represents a second line of defense to maintain water balance. Water balance disorders arise because of deficiency, resistance or inappropriate secretion of AVP or disturbances in thirst sensation (hypodipsia, polydipsia). People with water balance disorders are prone to develop hyponatremia or hypernatremia, which expose cells to osmotic stress and activate cell volume regulation mechanisms. This review covers several recent insights that have expanded our understanding of central osmoregulation, AQP2 regulation and cell volume regulation. This includes the role of with-no-lysine kinase 1 (WNK1) as a putative central osmolality sensor and, more generally, as an intracellular crowding sensor that coordinates the cell volume rescue response by activating sodium and potassium cotransporters. Furthermore, several new regulators of AQP2 have been identified, including for AVP-dependent AQP2 regulation (yes-associated protein, nuclear factor of activated T-cells, microRNAs) and AVP-independent AQP2 regulation (epidermal growth factor receptor, fluconazole, prostaglandin E2). It is also becoming increasingly clear that long-term cell volume adaptation to chronic hypotonicity through release of organic osmolytes comes at the expense of compromised organ function. This potentially explains the complications of chronic hyponatremia, including cognitive impairment, bone loss and vascular calcification. This review will illustrate why these new insights derived from basic science are also relevant for developing new approaches to treat water balance disorders.

Chemokine-mediated F-actin dynamics, polarity, and migration in B lymphocytes depend on WNK1 signaling.

Ligand-engaged chemokine receptors trigger nucleotide exchange in heterotrimeric Gαi proteins, which stimulates cytoskeletal reorganization and cell polarity changes. To better understand the signaling events responsible for these cellular changes, we focused on early changes in F-actin dynamics after engagement of the chemokine receptor CXCR5 in murine splenic B cells. Within 10 seconds of exposure to the CXCR5 ligand CXCL13, three-dimensional lamellar-like pseudopods and F-actin-rich ridges appeared. The transient F-actin increase depended on Gαi2/3 signaling, the PI3K/AKT pathway, ERK activation, phospholipase C activity, and Rac1/2 activation mediated by Dock2 (dedicator of cytokinesis 2). Immunoblot analyses identified the kinase WNK1 (with no lysine kinase 1) as a potential early AKT effector. Treating B cells with specific WNK inhibitors disrupted F-actin dynamics and impaired B cell polarity, motility, and chemotaxis. These changes were mimicked in a murine B cell line by CRISPR-Cas9 gene editing of Wnk1, which also suggested that WNK1 contributed to B cell proliferation. Administration of a single dose of a WNK inhibitor transiently reduced B cell motility and polarity in the lymph nodes of live mice. These results indicate that WNK1 signaling maintains B cell responsiveness to CXCL13 and suggest that pharmacological inhibition of WNK1, which is involved in cancer progression and blood pressure regulation, may affect humoral immunity.

WNK1 mediates amphiregulin-induced MMP9 expression and cell invasion in human extravillous trophoblast cells

The invasion of human extravillous trophoblast (EVT) cells is a critical event required for a successful pregnancy. Amphiregulin, a ligand of the epidermal growth factor receptor (EGFR), has been shown to stimulate cell invasion in an immortalized human EVT cell line, HTR-8/SVneo. The with-no-lysine kinase 1 (WNK1) is involved in regulating cell invasion. It is known that WNK1 is expressed in the human placenta, but its role in human EVT cells remains unknown. In the present study, we show that AREG treatment phosphorylated WNK1 at Thr60 in both HTR-8/SVneo and primary human EVT cells. The stimulatory effect of AREG on WNK1 phosphorylation was mediated by the activation of PI3K/AKT, but not the ERK1/2 signaling pathway. AREG upregulated matrix metalloproteinase 9 (MMP9) but not MMP2. In addition, cell invasiveness was increased in response to the treatment of AREG. Using the siRNA-mediated knockdown approach, our results showed that the knockdown of WNK1 attenuated the AREG-induced upregulation of MMP9 expression and cell invasion. Moreover, the expression of WNK1 was downregulated in the placentas with preeclampsia, a disease resulting from insufficiency of EVT cell invasion during pregnancy. This study discovers the physiological function of WNK1 in human EVT cells and provides important insights into the regulation of MMP9 and cell invasion in human EVT cells.

Maternal taurine as a modulator of Cl- homeostasis as well as of glycine/GABAA receptors for neocortical development.

During brain and spinal cord development, GABA and glycine, the inhibitory neurotransmitters, cause depolarization instead of hyperpolarization in adults. Since glycine and GABAA receptors (GABAARs) are chloride (Cl-) ion channel receptor, the conversion of GABA/glycine actions during development is influenced by changes in the transmembrane Cl- gradient, which is regulated by Cl- transporters, NKCC1 (absorption) and KCC2 (expulsion). In immature neurons, inhibitory neurotransmitters are released in a non-vesicular/non-synaptic manner, transitioning to vesicular/synaptic release as the neuron matures. In other word, in immature neurons, neurotransmitters generally act tonically. Thus, the glycine/GABA system is a developmentally multimodal system that is required for neurogenesis, differentiation, migration, and synaptogenesis. The endogenous agonists for these receptors are not fully understood, we address taurine. In this review, we will discuss about the properties and function of taurine during development of neocortex. Taurine cannot be synthesized by fetuses or neonates, and is transferred from maternal blood through the placenta or maternal milk ingestion. In developing neocortex, taurine level is higher than GABA level, and taurine tonically activates GABAARs to control radial migration as a stop signal. In the marginal zone (MZ) of the developing neocortex, endogenous taurine modulates the spread of excitatory synaptic transmission, activating glycine receptors (GlyRs) as an endogenous agonist. Thus, taurine affects information processing and crucial developmental processes such as axonal growth, cell migration, and lamination in the developing cerebral cortex. Additionally, we also refer to the possible mechanism of taurine-regulating Cl- homeostasis. External taurine is uptake by taurine transporter (TauT) and regulates NKCC1 and KCC2 mediated by intracellular signaling pathway, with-no-lysine kinase 1 (WNK1) and its subsequent kinases STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Through the regulation of NKCC1 and KCC2, mediated by the WNK-SPAK/OSR1 signaling pathway, taurine plays a role in maintaining Cl- homeostasis during normal brain development.

Loss of endothelial CFTR drives barrier failure and edema formation in lung infection and can be targeted by CFTR potentiation

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS). Here, we identified loss of endothelial cystic fibrosis transmembrane conductance regulator (CFTR) as an important pathomechanism leading to lung barrier failure in pneumonia-induced ARDS. CFTR was down-regulated after Streptococcus pneumoniae infection ex vivo or in vivo in human or murine lung tissue, respectively. Analysis of isolated perfused rat lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular chloride ion and calcium ion concentrations ([Cl-]i and [Ca2+]i). Inhibition of the chloride ion-sensitive with-no-lysine kinase 1 (WNK1) protein with tyrphostin 47 or WNK463 replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+]i, whereas WNK1 activation by temozolomide attenuated it. Endothelial [Ca2+]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4-/-) developed less lung edema and protein leak than their wild-type littermates after infection with S. pneumoniae. The CFTR potentiator ivacaftor prevented lung CFTR loss, edema, and protein leak after S. pneumoniae infection in wild-type mice. In conclusion, lung infection caused loss of CFTR that promoted lung edema formation through intracellular chloride ion accumulation, inhibition of WNK1, and subsequent disinhibition of TRPV4, resulting in endothelial calcium ion influx and vascular barrier failure. Ivacaftor prevented CFTR loss in the lungs of mice with pneumonia and may, therefore, represent a possible therapeutic strategy in people suffering from ARDS due to severe pneumonia.

WNK1/HSN2 mediates neurite outgrowth and differentiation via a OSR1/GSK3β-LHX8 pathway

With no lysine kinase 1 (WNK1) phosphorylates and activates STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1) to regulate ion homeostasis in the kidney. Mutations in WNK1 result in dysregulation of the WNK1-SPAK/OSR1 pathway and cause pseudohypoaldosteronism type II (PHAII), a form of hypertension. WNK1 is also involved in the autosomal recessive neuropathy, hereditary sensory and autonomic neuropathy type II (HSANII). Mutations in a neural-specific splice variant of WNK1 (HSN2) cause HSANII. However, the mechanisms underlying HSN2 regulation in neurons and effects of HSN2 mutants remain unclear. Here, we found that HSN2 regulated neurite outgrowth through OSR1 activation and glycogen synthase kinase 3β (GSK3β). Moreover, HSN2-OSR1 and HSN2-GSK3β signalling induced expression of LIM homeobox 8 (Lhx8), which is a key regulator of cholinergic neural function. The HSN2-OSR1/GSK3β-LHX8 pathway is therefore important for neurite outgrowth. Consistently, HSN2 mutants reported in HSANII patients suppressed SPAK and OSR1 activation and LHX8 induction. Interestingly, HSN2 mutants also suppressed neurite outgrowth by preventing interaction of between wild-type HSN2 and GSK3β. These results indicate that HSN2 mutants cause dysregulation of neurite outgrowth via GSK3β in the HSN2 and/or WNK1 pathways.

Abstract LB506: Structure based small molecule drug design for targeting WNK1 in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous group of tumors lacking estrogen, progesterone, and HER-2 receptors. It accounts for 10-20% of breast cancers, appears in younger women, metastasizes fast, and is associated with poor prognosis and poor patient survival. There are no available tailored therapies for TNBC to date. One of the ways to treat TNBC is by targeting its metastasis feature. With-no-lysine kinase 1 (WNK1), which is known to interact with diverse signaling pathways including ion homeostasis, has been recently identified as one of 6 driver genes in a murine TNBC model. Published and preliminary data support the premise that WNK1 promotes epithelial-mesenchymal transition (EMT) and invasion in TNBC. Genetic and pharmacological inhibition of WNK1 blocks metastasis in a TNBC mouse model. Given the existence of four WNK isoforms in which kinase domains are 90% identical, developing a clinically available WNK1-specific inhibitor is an unmet need. We identified WNK1-selective inhibitors by a High Throughput Screen (HTS) of over 200,000 structurally diverse small molecules. A class of quinoline-derived compounds showed high specificity and potency for WNK1. A combination of molecular modeling, crystallography, and synthesis-based strategies were employed to improve the potency of compounds. Interestingly, the compound SC107-01 reported below 100 nM IC50 for WNK1 by in vitro assays. Also, it shows higher specificity for WNK1 over other kinases as evident by kinome-specificity profiling. We had previously crystallized the kinase domain of WNK1. The structure revealed its unique active site. Co-crystal structure of SC107-01 with WNK1 shows that the inhibitor binds to the WNKs’ specific active site and forms a unique set of interactions. Further modifications to the SC107-01 are underway to improve bioavailability and efficacy. In vivo toxicology and drug pharmacokinetics will be conducted in mouse models. We anticipate that this work will lay the foundation for developing a small molecule that inhibits WNK1 to combat metastasis in TNBC. Citation Format: Ashari Rashmi Kannangara, Melanie Rodriguez, Julita Chlebowicz, Radha Akella, Melanie H. Cobb, Uttam Tambar, Elizabeth J. Goldsmith. Structure based small molecule drug design for targeting WNK1 in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB506.

Loss of Endothelial CFTR Drives Barrier Failure and Edema Formation in Lung Infection and Can Be Targeted by CFTR Potentiation

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS), a potentially fatal lung disease characterized by hyperinflammation and endothelial barrier failure. Infectious and inflammatory stimuli can cause rapid downregulation of cystic fibrosis transmembrane conductance regulator (CFTR), and inhibition of CFTR has been proposed to increase lung microvascular endothelial permeability in vitro. Here, we identify loss of lung microvascular endothelial CFTR as important pathomechanism in lung barrier failure in pneumonia‐induced ARDS, delineate the molecular signaling pathway underlying this effect and identify CFTR potentiation as novel therapeutic strategy in ARDS.CFTR was downregulated in endothelial cells following Streptococcus pneumoniae(S.pn.) infection in human and murine lung tissue. Isolated perfused lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular Cl‐ and Ca2+concentrations ([Cl‐]i, [Ca2+]i). Inhibition of the Cl‐ sensitive with‐no‐lysine kinase 1 (WNK1) replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+]i while WNK1 activation attenuated it. Endothelial [Ca2+]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4‐/‐) developed less lung edema and protein leak than their wild‐type littermates following infection with S. pn. Conversely, lungs of heterozygous Wnk1‐deficient mice (Wnk1+/‑) showed spontaneous leak. The CFTR potentiator ivacaftor prevented CFTR loss and reduced endothelial leak in response to pneumolysin (PLY), a key S.pn. virulence factor, or plasma from COVID‐19 patients in vitro, and prevented lung edema and protein leak after S. pn.infection in vivo.Lung infection causes rapid loss of CFTR that promotes lung edema formation through intracellular Cl‐accumulation, inhibition of WNK1 and subsequent disinhibition of TRPV4, resulting in endothelial Ca2+ influx and vascular barrier failure. Ivacaftor prevents CFTR loss and may thus present a promising therapeutic strategy in ARDS due to severe pneumonia including COVID‐19.

With No Lysine Kinase 1 Promotes Metabolic Derangements and RV Dysfunction in Pulmonary Arterial Hypertension

Small molecule inhibition of with no lysine kinase 1 (WNK1) (WNK463) signaling activates adenosine monophosphate-activated protein kinase signaling and mitigates membrane enrichment of glucose transporters 1 and 4, which decreases protein O-GlcNAcylation and glycation. Quantitative proteomics of right ventricular (RV) mitochondrial enrichments shows WNK463 prevents down-regulation of several mitochondrial metabolic enzymes. and metabolomics analysis suggests multiple metabolic processes are corrected. Physiologically, WNK463 augments RV systolic and diastolic function independent of pulmonary arterial hypertension severity. Hypochloremia, a condition of predicted WNK1 activation in patients with pulmonary arterial hypertension, is associated with more severe RV dysfunction. These results suggest WNK1 may be a druggable target to combatmetabolic dysregulation and may improve RV function and survival in pulmonary arterial hypertension.

Human adenylate kinase 6 regulates WNK1 (with no lysine kinase-1) phosphorylation states and affects ion homeostasis in NT2 cells

Adenylate kinase 6 (AK6), a nucleus localized phosphotransferase in mammalians, shows ubiquitously expression and broad substrate activity in different tissues and cell types. Although the function of AK6 has been extensively studied in different cancer cell lines, its role in mammalian germline is still unknown. Here we showed that knockdown of AK6 inhibits cell proliferation and promotes cell apoptosis in human testicular carcinoma (NT2 cells). Co-immunoprecipitation experiment and in vitro pull down assay identified WNK1 (with no lysine kinase-1) as one of the AK6 interacting proteins in NT2 cells. Moreover, we found that AK6 regulates the phosphorylation states of WNK1 (Thr60) and affects phosphorylation level of Akt (Ser473) upon hypotonic condition, probably affecting chloride channel and regulating ion transport and homeostasis in NT2 cells and consequently contributing to the decreased cell proliferation rate. In conclusion, AK6 regulates WNK1 phosphorylation states and affects ion homeostasis in NT2 cells. These findings provide new insights into the function of AK6 and WNK1 in human testicular carcinoma. This work also provides foundation for further mechanism study of AK6 in spermatogenesis.

Characterization of chemical components with diuretic potential from Pyrrosia petiolosa

Three compounds with diuretic potential were identified from the 95% ethanol extract of Pyrrosia petiolosa (Christ) Ching. Among them, one was a new benzanilide named petiolide A (1), and the other two were phenolic derivatives barbatic acid (2) and kaempferol (3). Their structures were elucidated based on extensive spectral analyses and comparison with the literature data. The docking experiments of all compounds into the active site of the With-No-Lysine kinase 1 (WNK1) domain demonstrated that kaempferol (3) was the most effective component with diuretic potential for its comparative diuretic effect to that of an orally bioavailable WNK inhibitor WNK463 (docking score −10.99 vs −11.09).

The WNK signaling pathway and salt-sensitive hypertension

The distal nephron of the kidney has a central role in sodium and fluid homeostasis, and disruption of this homeostasis due to mutations of with-no-lysine kinase 1 (WNK1), WNK4, Kelch-like 3 (KLHL3), or Cullin 3 (CUL3) causes pseudohypoaldosteronism type II (PHAII), an inherited hypertensive disease. WNK1 and WNK4 activate the NaCl cotransporter (NCC) at the distal convoluted tubule through oxidative stress-responsive gene 1 (OSR1)/Ste20-related proline-alanine-rich kinase (SPAK), constituting the WNK-OSR1/SPAK-NCC phosphorylation cascade. The level of WNK protein is regulated through degradation by the CUL3-KLHL3 E3 ligase complex. In the normal state, the activity of WNK signaling in the kidney is physiologically regulated by sodium intake to maintain sodium homeostasis in the body. In patients with PHAII, however, because of the defective degradation of WNK kinases, NCC is constitutively active and not properly suppressed by a high salt diet, leading to abnormally increased salt reabsorption and salt-sensitive hypertension. Importantly, recent studies have demonstrated that potassium intake, insulin, and TNFα are also physiological regulators of WNK signaling, suggesting that they contribute to the salt-sensitive hypertension associated with a low potassium diet, metabolic syndrome, and chronic kidney disease, respectively. Moreover, emerging evidence suggests that WNK signaling also has some unique roles in metabolic, cardiovascular, and immunological organs. Here, we review the recent literature and discuss the molecular mechanisms of the WNK signaling pathway and its potential as a therapeutic target.

Role of WNK1/OSR1 in maintaining endothelial tight junction integrity

Deletion of the WNK1 (with no lysine kinase‐1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial‐specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive‐1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to regulate TGF‐β/Smad signaling which induces epithelial‐mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF‐βII receptor at tight junctions which mediates dissolution of tight junction upon activation by TGF‐β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for TGF‐β/Smad‐induced disruption of cell‐cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 pharmacological inhibition decreased the expression of TGF‐β‐induced EMT mediators such as the receptor tyrosine kinase Axl which is known to be important for maintaining tight junction integrity in HDMECs (Human Dermal Microvascular Endothelial Cells) (Normalized densitometry values; WNKi‐ Axl: 0.2 ± 0.05 vs. siControl: 1, n=3) and in primary HUVECs (Human Umbilical Vein Endothelial Cells) (normalized densitometry values; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n=3, *p<0.05). We also determined that OSR1 and Occludin, a tight junction protein are co‐localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co‐localization and tight junction breakdown during TGF‐β signaling in primary HUVECs (Pearson’s coefficient; Control; 0.08 ± 0.2 vs. TGF‐β: 0.61 ± 0.15 vs. TGF‐β+WNK1i: 0.12 ± 0.25, n=2, 2 replicates per experiment). Finally, we observed that bands corresponding to phosphorylated form of Occludin on a Western blot were enhanced in WNK inhibited samples compared to control (normalized densitometry values; TGF‐β+WNKi: 1 vs. TGF‐β: 0.21 ± 0.07, n=2). Since Occludin is known to be dephosphorylated during tight junction breakdown, the presence of the phosphorylated form of Occludin with WNK inhibition may indicate the disruption of cellular processes leading to TGF‐β‐dependent tight junction breakdown. These data suggest WNK1‐dependent function of OSR1 in cell migration and angiogenesis through regulating tight junction integrity. Combined, these data indicate the importance of WNK1 function in endothelial cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis as well as cancer.Support or Funding InformationAmerican Heart Association postdoctoral fellowship to Ankita Jaykumar and NIH funding R01 HL147661 to Melanie H. Cobb

Regulation of PI3k-WNK Pathway by Ethyl Acetate Partition Fraction of Gardenia jasminoides var. radicans Makino in SHR, NRK52e Cells, and IMCD3 Cells

The antihypertensive mechanism was studied of the ethyl acetate fraction of the ethanol extract (EAPF) of Gardenia jasminoides var. radicans Makino (GJRM). GJRM is a fake product of Gardenia jasminoides Ellis ( G. jasminoides), but in China’s Henan province, the production of GJRM is much more than G. jasminoides’s, but its traditional usage is as a dye. Gardenia jasminoides can be used to reduce blood pressure and blood glucose levels. The chemical compositions of GJRM and G. jasminoides are similar, and we previously confirmed that GJRM can also reduce blood pressure. Here, we report that the EAPF of GJRM could activate the phosphoinositide 3-kinases (PI3K) pathway in the kidneys of spontaneously hypertensive rats, thus increasing the content of nitric oxide and bradykinin in sera and decreasing endothelin-1 content. EAPF can also decrease the levels of with-no-lysine kinase 1 (WNK1) expression, WNK4 and oxidative stress-responsive kinase 1 messenger ribonucleic acid (mRNA), and Na-K-2Cl cotransporter and sodium chloride cotransporters mRNA and phosphorylation. To investigate the antihypertensive effects of the EAPF of GJRM, 5 monoterpenoids isolated from EAPF were studied for their effects on NRK52e and IMCD3 cells. These compounds inhibited the PI3K-WNK signaling pathway to varying degrees under hypotonic conditions; 4-methoxyrehmapicrogenin had the best effect.

Regulation of erythrocyte Na+/K+/2Cl− cotransport by an oxygen-switched kinase cascade

Many erythrocyte processes and pathways, including glycolysis, the pentose phosphate pathway (PPP), KCl cotransport, ATP release, Na+/K+-ATPase activity, ankyrin-band 3 interactions, and nitric oxide (NO) release, are regulated by changes in O2 pressure that occur as a red blood cell (RBC) transits between the lungs and tissues. The O2 dependence of glycolysis, PPP, and ankyrin-band 3 interactions (affecting RBC rheology) are controlled by O2-dependent competition between deoxyhemoglobin (deoxyHb), but not oxyhemoglobin (oxyHb), and other proteins for band 3. We undertook the present study to determine whether the O2 dependence of Na+/K+/2Cl- cotransport (catalyzed by Na+/K+/2Cl- cotransporter 1 [NKCC1]) might similarly originate from competition between deoxyHb and a protein involved in NKCC1 regulation for a common binding site on band 3. Using three transgenic mouse strains having mutated deoxyhemoglobin-binding sites on band 3, we found that docking of deoxyhemoglobin at the N terminus of band 3 displaces the protein with no lysine kinase 1 (WNK1) from its overlapping binding site on band 3. This displacement enabled WNK1 to phosphorylate oxidative stress-responsive kinase 1 (OSR1), which, in turn, phosphorylated and activated NKCC1. Under normal solution conditions, the NKCC1 activation increased RBC volume and thereby induced changes in RBC rheology. Because the deoxyhemoglobin-mediated WNK1 displacement from band 3 in this O2 regulation pathway may also occur in the regulation of other O2-regulated ion transporters, we hypothesize that the NKCC1-mediated regulatory mechanism may represent a general pattern of O2 modulation of ion transporters in erythrocytes.

The function of WNK1/OSR1 in cell migration and angiogenesis

Deletion of the WNK1 (with no lysine kinase-1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial-specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive-1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to be stimulated by TGF-β/Smad signaling which induces epithelial-mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF-βII receptor at tight junctions which mediates ubiquitination of GTP-RhoA and dissolution of tight junction upon activation by TGF-β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for the expression of TGF-β/Smad-induced EMT factors, suppression of RhoA activity and disruption of cell-cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 siRNA-mediated knockdown decreased the expression of TGF-β-induced EMT mediators such as Slug, the receptor tyrosine kinase Axl and the transcription factor p63 (ΔNp63α) (normalized densitometry values to control; siWNK1-Slug: 0.5 ± 0.25, Axl: 0.2 ± 0.05, p63: 0.35 ± 0.15 vs. siControl: 1, n = 2) in MCFDCIS cancer cell line. Similarly, WNK1 pharmacological inhibition decreased Axl expression (normalized densitometry values to control; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n = 3, *p<0.05) in primary HUVECs (Human Umbilical Vein Endothelial Cells). This suggests that WNK1 is involved in Slug/p63/Axl network in endothelial and cancer cells. We found that knockdown of WNK1 increased F-actin content (normalized arbitrary fluorescence units to control; siWNK1: 1.6 ± 0.3 vs. siControl: 1, n = 2, 4–5 replicates per experiment) in HeLa cells. Because RhoA activity regulates actin dynamics, RhoA activity was measured in WNK1 depleted cells and the ratio of GTP-bound RhoA to total RhoA was found to be significantly increased (siWNK1: 3.08 ± 0.75 vs. siControl: 0.34 ± 0.05, n = 3, *p<0.035). Finally, we determined that OSR1 and Occludin, a tight junction protein are co-localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co-localization during TGF-β signaling in primary HUVECs (Pearson's coefficient; Control: 0.08 ± 0.2 vs. TGF-β: 0.61 ± 0.15 vs. TGF-β+WNK1i: 0.12 ± 0.25, n = 2, 2 replicates per experiment). These data suggest WNK1-dependent function of OSR1 in cell migration and angiogenesis through effects on GTP-RhoA degradation. Combined, these data indicate the importance of WNK1 function in endothelial and cancer cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis, ischemia-reperfusion injury as well as cancer. Support or Funding Information Early part of this work was supported by NIH 5R01GM053032-16 grant. Current work is being supported by the Welch Foundation grant I-1243. Co-localization of OSR1 and Occludin in primary HUVECs This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Akt3 kinase suppresses pinocytosis of low-density lipoprotein by macrophages via a novel WNK/SGK1/Cdc42 protein pathway

Fluid-phase pinocytosis of LDL by macrophages is regarded as a novel promising target to reduce macrophage cholesterol accumulation in atherosclerotic lesions. The mechanisms of regulation of fluid-phase pinocytosis in macrophages and, specifically, the role of Akt kinases are poorly understood. We have found previously that increased lipoprotein uptake via the receptor-independent process in Akt3 kinase-deficient macrophages contributes to increased atherosclerosis in Akt3-/- mice. The mechanism by which Akt3 deficiency promotes lipoprotein uptake in macrophages is unknown. We now report that Akt3 constitutively suppresses macropinocytosis in macrophages through a novel WNK1/SGK1/Cdc42 pathway. Mechanistic studies have demonstrated that the lack of Akt3 expression in murine and human macrophages results in increased expression of with-no-lysine kinase 1 (WNK1), which, in turn, leads to increased activity of serum and glucocorticoid-inducible kinase 1 (SGK1). SGK1 promotes expression of the Rho family GTPase Cdc42, a positive regulator of actin assembly, cell polarization, and pinocytosis. Individual suppression of WNK1 expression, SGK1, or Cdc42 activity in Akt3-deficient macrophages rescued the phenotype. These results demonstrate that Akt3 is a specific negative regulator of macropinocytosis in macrophages.

Impaired degradation of medullary WNK4 in the kidneys of KLHL2 knockout mice

Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, Kelch-like 3 (KLHL3), and Cullin3 (CUL3) genes were identified as being responsible for hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). Normally, the KLHL3/CUL3 ubiquitin ligase complex degrades WNKs. In PHAII, the loss of interaction between KLHL3 and WNK4 increases levels of WNKs because of impaired ubiquitination, leading to abnormal over-activation of the WNK-OSR1/SPAK-NCC cascade in the kidney's distal convoluted tubules (DCT). KLHL2, which is highly homologous to KLHL3, was reported to ubiquitinate and degrade WNKs in vitro. Mutations in KLHL2 have not been reported in patients with PHAII, suggesting that KLHL2 plays a different physiological role than that played by KLHL3 in the kidney. To investigate the physiological roles of KLHL2 in the kidney, we generated KLHL2−/− mice. KLHL2−/− mice did not exhibit increased phosphorylation of the OSR1/SPAK-NCC cascade and PHAII-like phenotype. KLHL2 was predominantly expressed in the medulla compared with the cortex. Accordingly, medullary WNK4 protein levels were significantly increased in the kidneys of KLHL2−/− mice. KLHL2 is indeed a physiological regulator of WNK4 in vivo; however, its function might be different from that of KLHL3 because KLHL2 mainly localized in medulla.

KLHL3 Knockout Mice Reveal the Physiological Role of KLHL3 and the Pathophysiology of Pseudohypoaldosteronism Type II Caused by Mutant KLHL3.

Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, kelch-like 3 (KLHL3), and cullin3 (CUL3) genes are known to cause the hereditary disease pseudohypoaldosteronism type II (PHAII). It was recently demonstrated that this results from the defective degradation of WNK1 and WNK4 by the KLHL3/CUL3 ubiquitin ligase complex. However, the other physiological in vivo roles of KLHL3 remain unclear. Therefore, here we generated KLHL3-/- mice that expressed β-galactosidase (β-Gal) under the control of the endogenous KLHL3 promoter. Immunoblots of β-Gal and LacZ staining revealed that KLHL3 was expressed in some organs, such as brain. However, the expression levels of WNK kinases were not increased in any of these organs other than the kidney, where WNK1 and WNK4 increased in KLHL3-/- mice but not in KLHL3+/- mice. KLHL3-/- mice also showed PHAII-like phenotypes, whereas KLHL3+/- mice did not. This clearly demonstrates that the heterozygous deletion of KLHL3 was not sufficient to cause PHAII, indicating that autosomal dominant type PHAII is caused by the dominant negative effect of mutant KLHL3. We further demonstrated that the dimerization of KLHL3 can explain this dominant negative effect. These findings could help us to further understand the physiological roles of KLHL3 and the pathophysiology of PHAII caused by mutant KLHL3.