Ste20-related Kinase Research Articles

Hematopoietic Progenitor Kinase 1 in Tumor Immunology: A Medicinal Chemistry Perspective.

Hematopoietic progenitor kinase 1 (HPK1), a hematopoietic cell-restricted member of the serine/threonine Ste20-related protein kinases, is a negative regulator of the Tcell receptor, B cell receptor, and dendritic cells. Loss of HPK1 kinase function increases cytokine secretion and enhances Tcell signaling, virus clearance, and tumor growth inhibition. Therefore, HPK1 is considered a promising target for tumor immunotherapy. Several HPK1 inhibitors have been reported to regulate Tcell function. In addition, HPK1-targeting PROTACs, which can induce the degradation of HPK1, have also been developed. Here, we provide an overview of research concerning HPK1 protein structure, function, and inhibitors and propose perspectives and insights for the future development of agents targeting HPK1.

Abstract P241: A highly potent HPK1 inhibitor augments immune cell activation and anti-tumor immunity in a syngeneic tumor mouse model

Abstract Background: HPK1 (MAP4K1) is a serine/threonine Ste20-related protein kinase that belongs to the mitogen-activated protein kinase (MAPK) family. HPK1 is mainly expressed in hematopoietic cells and serves as a negative regulator of anti-tumor immunity through modulating the activation of lymphocytes and dendritic cells. Upon TCR activation, HPK1 phosphorylates the adaptor protein SLP76 at Ser376 to destabilize the SLP76 microclusters, which leads to the attenuation of the TCR signaling. The reported anti-tumor efficacy data from HPK1 knockout and kinase-dead knock-in mouse models support HPK1 as a novel intracellular I/O target. Methods: Regor CARD platform (Computer Accelerated Rational Discovery) was deployed to identify potent and selective inhibitors of HPK1. Biochemical assays and primary human pan T cell-based cellular assays were utilized to support the structure-activity relationship (SAR) analysis and inhibitor optimization. In vitro and in vivo target engagement studies were conducted in Jurkat T cells and mouse splenocytes, respectively. In vivo efficacy study data were generated using CT-26 syngeneic tumor mouse model. Results: Highly potent HPK1 inhibitors with good selectivity against the liable immune kinases were identified. RGT-197, one of the lead inhibitors demonstrated a significant increase of IL-2 secretion in primary human pan T cells upon TCR activation. It inhibited TCR-induced phosphorylation of SLP76 at Ser-376 in vitro and in vivo. Anti-tumor efficacy was observed in CT-26 tumor-bearing mice by oral dosing of RGT-197 as a single agent and in combination with an anti-PD1-monoclonal antibody. Furthermore, RGT-197 increased the level of cytokines important for anti-tumor immunity in vivo. Conclusion: RGT-197, a potent and selective HPK1 inhibitor, provides a potential opportunity as a small molecule I/O agent to boost anti-tumor immunity either as monotherapy or in combination with immune checkpoint inhibitors. Citation Format: Hao Liu, Lei Wu, Song Feng, Wei Huang, Huijuan Li, Jing Lin, Yangyang Liu, Liufeng Mei, Baoqi Ren, Julie Xie, Lili Yao, Wenge Zhong. A highly potent HPK1 inhibitor augments immune cell activation and anti-tumor immunity in a syngeneic tumor mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P241.

Calcium-Sensing Receptor and Regulation of WNK Kinases in the Kidney.

The kidney is essential for systemic calcium homeostasis. Urinary calcium excretion can be viewed as an integrative renal response to endocrine and local stimuli. The extracellular calcium-sensing receptor (CaSR) elicits a number of adaptive reactions to increased plasma Ca2+ levels including the control of parathyroid hormone release and regulation of the renal calcium handling. Calcium reabsorption in the distal nephron of the kidney is functionally coupled to sodium transport. Apart from Ca2+ transport systems, CaSR signaling affects relevant distal Na+-(K+)-2Cl− cotransporters, NKCC2 and NCC. NKCC2 and NCC are activated by a kinase cascade comprising with-no-lysine [K] kinases (WNKs) and two homologous Ste20-related kinases, SPAK and OSR1. Gain-of-function mutations within the WNK-SPAK/OSR1-NKCC2/NCC pathway lead to renal salt retention and hypertension, whereas loss-of-function mutations have been associated with salt-losing tubulopathies such as Bartter or Gitelman syndromes. A Bartter-like syndrome has been also described in patients carrying gain-of-function mutations in the CaSR gene. Recent work suggested that CaSR signals via the WNK-SPAK/OSR1 cascade to modulate salt reabsorption along the distal nephron. The review presented here summarizes the latest progress in understanding of functional interactions between CaSR and WNKs and their potential impact on the renal salt handling and blood pressure.

Multimodal Analysis of STRADA Function in Brain Development.

mTORopathies are a heterogeneous group of neurological disorders characterized by malformations of cortical development (MCD), enhanced cellular mechanistic target of rapamycin (mTOR) signaling, and epilepsy that results from mutations in mTOR pathway regulatory genes. Homozygous mutations (del exon 9–13) in the pseudokinase STE20-related kinase adaptor alpha (STRAD-α; STRADA), an mTOR modulator, are associated with Pretzel Syndrome (PS), a neurodevelopmental disorder within the Old Order Mennonite Community characterized by megalencephaly, intellectual disability, and intractable epilepsy. To study the cellular mechanisms of STRADA loss, we generated CRISPR-edited Strada mouse N2a cells, a germline mouse Strada knockout (KO−/−) strain, and induced pluripotent stem cell (iPSC)-derived neurons from PS individuals harboring the STRADA founder mutation. Strada KO in vitro leads to enhanced mTOR signaling and iPSC-derived neurons from PS individuals exhibit enhanced cell size and mTOR signaling activation, as well as subtle alterations in electrical firing properties e.g., increased input resistance, a more depolarized resting membrane potential, and decreased threshold for action potential (AP) generation. Strada−/− mice exhibit high rates of perinatal mortality and out of more than 100 litters yielding both WT and heterozygous pups, only eight Strada−/− animals survived past P5. Strada−/− mice are hypotonic and tremulous. Histopathological examination (n = 5 mice) revealed normal gross brain organization and lamination but all had ventriculomegaly. Ectopic neurons were seen in all five Strada−/− brains within the subcortical white matter mirroring what is observed in human PS brain tissue. These distinct experimental platforms demonstrate that STRADA modulates mTOR signaling and is a key regulator of cell size, neuronal excitability, and cortical lamination.

Cerebrospinal fluid of chronic osteoarthritic patients induced interleukin-6 release in human glial cell-line T98G

BackgroundChronic osteoarthritic pain is not well understood in terms of its pathophysiological mechanism. Activated glial cells are thought to play a role in the maintenance of chronic pain. T98G glioblastoma cell line was previously observed to release higher amounts of interleukin-6 (IL-6) when treated with cerebrospinal fluid (CSF) from patients with another chronic pain condition, post-herpetic neuralgia. In this study, we investigated the ability of CSF from patients diagnosed with knee osteoarthritis suffering from chronic pain, to trigger the release of pro-inflammatory cytokines, IL-6, IL-1beta and tumour necrosis factor alpha (TNF-α) from T98G. Characterization of upstream signalling was also explored.MethodsFifteen osteoarthritis patients undergoing total knee replacement due to chronic knee pain and 15 patients without pain undergoing other surgeries with spinal anaesthesia were prospectively recruited. CSF was collected during anaesthesia. CSF were added to cultured T98G cells in the presence of lipopolysaccharide. IL-6, IL-1β and TNF-α release from T98G cells were measured using enzyme immunoassay. Antibody array and western blotting were performed using CSF-triggered T98G cell lysates to identify possible signalling targets. Age, gender and pain scores were recorded. Mann-Whitney U test was used to compare IL-6 release and protein expression between groups. Association between IL-6 and pain score was analysed using linear regression.ResultsSignificant higher levels of IL-6 were released by T98G cells when induced by osteoarthritis patients’ CSF in the presence of LPS. The IL-6 levels showed positive association with pain score (adjusted B estimate = 10.1 (95% Confidence Interval 4.3–15.9); p = 0.001). Antibody array conducted with 6 pooled T98G cell lysate induced with osteoarthritis pain patient CSF identified greater than 2-fold proteins including STE20-related kinase adaptor protein and spleen tyrosine kinase. Further validation done using western blotting of individual CSF-triggered T98G cell lysate showed non-significant increase.ConclusionHigher IL-6 release from T98G when triggered by OA-CSF, in the presence of LPS, suggest the presence of “unknown molecule” in CSF that may be crucial in the maintenance phase of chronic pain in our osteoarthritis population. Further studies on the signalling pathways involved in pain and relevance of IL-6 release from T98G cells in other pain models are needed.

A Novel Na + -K + -Cl − Cotransporter 1 Inhibitor STS66* Reduces Brain Damage in Mice After Ischemic Stroke

Background and Purpose- Inhibition of brain NKCC1 (Na+-K+-Cl- cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods- Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)-induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results- Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II-induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions- The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

MST3 (mammalian Ste20-like protein kinase 3), a novel gene involved in ion homeostasis and renal regulation of blood pressure in spontaneous hypertensive rats.

Defective renal salt and water excretion, together with increased salt intake, frequently contributes to hypertension. Recent studies indicate that Ste20 family kinases, such as proline-alanine-rich Ste20-related kinase (SPAK) and oxidative stress-response protein 1 (OSR1), are regulators of cell volume, ion transport, and hypertension. The aim of this study was to investigate whether mammalian sterile 20-like protein kinase 3 (MST3), which is also a stress-regulated kinase, is involved in the development of hypertension. MST3 expression was compared in Wistar-Kyoto (WKY) and spontaneously hypertensive rat (SHR) kidneys. MST3 expression was markedly reduced in principal cells of the collecting ducts from the renal inner medulla of SHR. The downregulation of MST3 expression was observed before and after the onset of hypertension in SHR. Mice fed high-salt diets (HS) exhibited a significant increase in MST3 protein level. This is the first study reporting that MST3, a Ste20-like kinase, exerts a conserved regulatory role in sodium homeostasis after high-salt diet and in the development of hypertension.

Novel Homozygous Deletion in STRADA Gene Associated With Polyhydramnios, Megalencephaly, and Epilepsy in 2 Siblings: Implications for Diagnosis and Treatment.

Mutations in the STE20-related kinase adaptor α ( STRADA) gene have been reported to cause an autosomal recessive neurodevelopmental disorder characterized by infantile-onset epilepsy, developmental delay, and craniofacial dysmorphisms. To date, there have been 17 reported individuals diagnosed with STRADA mutations, 16 of which are from a single Old Order Mennonite cohort and share a deletion of exons 9-13. The remaining individual is of consanguineous Indian descent and has a homozygous single-base pair duplication. We report a novel STRADA gene deletion of exons 7-9 in 2 sisters from nonconsanguineous parents, as well as an improvement in seizure control in 1 sibling following treatment with sirolimus, an m-Tor inhibitor of potential benefit to patients with this genetic mutation.

Metabolic stress directs WNK kinases to aggresomes for pooling and degradation in the distal convoluted tubule of the kidney

ObjectiveRenal salt retention due to excessive function of the renal NaCl cotransporter (NCC) along the distal convoluted tubule (DCT) plays a central role in the pathogenesis of hypertension. The Western type diet with high salt and low potassium content (HS/LK) has been shown to increase NCC activity by stimulating its phosphorylation via a kinase cascade comprising with‐no‐lysine kinases (WNKs) and a Ste20‐related kinase (SPAK). WNKs and SPAK have been shown to accumulate in perinuclear aggregates in response to potassium depletion, but the functional significance of these aggregates remained obscure. These structures may in part reflect a degradative pathway but may serve pooling functions as well.MethodsMice received HS/LK or control diets for 10 days. WNK1‐transfected DCT cells were exposed to vehicle, hyperosmotic stress, or potassium depletion. WNK1, WNK4, SPAK, phospho‐S383 SPAK, and various autophagy markers were studied by immunofluorescence and immunoblotting. Electron microscopy was applied for ultrastructural analysis.ResultsHS/LK diet induced accumulation of WNKs and SPAK in the form of perinuclear protein aggregates within DCT cells. These aggregates were rare in DCT of mice treated with control diet and virtually absent after administration of high potassium diet. The autophagy marker ATG5 was present in the aggregates and the 20S proteasome signal was intensified adjacently, whereas standard lysosomal markers showed no significant association with the punctate WNK/SPAK signal. Ultrastructural analysis of DCT in kidneys of mice treated with HS/LK revealed abundant aggresomes adjacent to rough endoplasmic reticulum and clathrin‐coated vesicles. Evaluation of cultured DCT cells transfected with WNK1 or KS‐WNK1 revealed formation of similar aggresomes in response to potassium depletion, whereas hyperosmotic stress induced formation of autophagosomes. Pretreatment of cells with proteasome inhibitor MG‐132 or autophagy/lysosome inhibitor bafilomycin A1 prior to potassium depletion substantially augmented the size of WNK1‐containing aggresomes.ConclusionChallenging of DCT epithelium induces the formation of perinuclear aggresomes which pool the components of the kinase cascade controlling NCC activity. From there, WNKs and SPAK may either be degraded via proteasomes or lysosomes, or reintroduced into the signal chain.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphorylation by PKC and PKA regulate the kinase activity and downstream signaling of WNK4

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.

Sodium-chloride cotransporter activity regulated by extracellular potassium

The thiazide-sensitive sodium-chloride cotransporter (NCC) is exclusively expressed in the apical membrane of the renal distal convoluted tubule (DCT), and it is important for maintaining fluid and salt balance. NCC is responsible for electroneutral NaCl reabsorption, and its activity is determined by phosphorylation, which was reported to be regulated by WNK-stimulated Ste20-type kinases, Ste20-related proline alanine-rich kinase, and oxidative stress response 1 kinase. WNK kinases have chloride-binding sites, and WNK4 has the highest sensitivity to intracellular chloride concentration ([Cl−]i) compared with WNK1 and WNK3. NCC dysfunction often comes together with abnormal urinary potassium excretion, which is not only highlighted by Mendelian disorders such as familial hyperkalemic hypertension (FHHt) and Gitelman syndrome but also presented more frequently by long-term usage of thiazides, specific inhibitors of NCC. Recent studies have shown that extracellular potassium (K+) can modulate DCT cell membrane voltage and in turn intracellular Cl−, which regulates phosphorylation of WNK kinases. Additional Cl− independent mechanisms were also reported by several groups. This paper is a brief review of the recent discoveries on mechanisms of NCC regulation by extracellular potassium.

The European Eel NCCβ Gene Encodes a Thiazide-resistant Na-Cl Cotransporter

The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCβ is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCβ from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCβ is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCβ is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCβ exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter.

The Core Molecular Machinery Used for Engulfment of Apoptotic Cells Regulates the JNK Pathway Mediating Axon Regeneration in Caenorhabditis elegans.

The mechanisms that govern the ability of specific neurons to regenerate their axons after injury are not well understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we identify two components functioning upstream of the JNK pathway: the Ste20-related protein kinase MAX-2 and the Rac-type GTPase CED-10. CED-10, when bound by GTP, interacts with MAX-2 and functions as its upstream regulator in axon regeneration. CED-10, in turn, is activated by axon injury via signals initiated from the integrin α-subunit INA-1 and the nonreceptor tyrosine kinase SRC-1 and transmitted via the signaling module CED-2/CrkII-CED-5/Dock180-CED-12/ELMO. This module is also known to regulate the engulfment of apoptotic cells during development. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. The molecular mechanisms of axon regeneration after injury remain poorly understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we show that integrin, Rac-GTPase, and several other molecules, all of which are known to regulate engulfment of apoptotic cells during development, also regulate axon regeneration. This signaling module activates the JNK-MAPK cascade via MAX-2, a PAK-like protein kinase that binds Rac. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway.

Abstract WP269: New Insights Into Worsened Cerebral Ischemia in Hypertensive Rats: Roles of WNK-SPAK/OSR1-NKCC1 Signaling

Background and Purpose: WNK kinases [with-no-lysine (K)], Ste20-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) are a family of evolutionarily conserved regulators of ion transporter proteins. The kinases alter the phosphorylation state of ion transporter proteins and so play an important role in renal salt handling and in the pathogenesis of hypertension. We previously reported that pharmacological inhibition or genetic knockout of Na+-K+-2Cl- cotransporter 1 (NKCC1) is neuroprotective against ischemic injury. Our current study investigated whether WNK-SPAK/OSR1-NKCC1 signaling is augmented in cerebral ischemia in hypertension. Methods: Cerebral ischemia was induced in adult male Wistar Kyoto (WKY) or spontaneously hypertensive rats (SHR) by 2-hour occlusion of the left middle cerebral artery (MCAO) followed by reperfusion. Brain tissues were harvested for immunoblot assessment of expression levels of NKCC1, SPAK/OSR1 or WNK1-4. Another two cohorts of WKY and SHR were treated with saline or NKCC1 inhibitor bumetanide (BMT, 10 mg/kg/day, i.p.) after MCAO. In these animals, ischemic infarct volume was determined by TTC staining and behavioral deficits were analyzed by neurological scoring and turning-alley test. Results: Basal levels of total NKCC1 protein were significantly higher in the contralateral (CL) cortical tissues in SHR brains than WKY brains. Ischemia was associated with increased phosphorylation of NKCC1 in the ipsilateral (IL) cortex of SHR, but not WKY at 6 hours post-ischemia. WNK1 increased 6.1±1.5 fold and WNK4 increased 8.7±1.7 fold in IL cortex of SHR, but not in that of WKY, and phosphorylated SPAK/OSR1 was unchanged. Lastly, sensitivity to pharmacological NKCC1 inhibition was higher in SHR than WKY rats. BMT blunted the MCAO-induced infarction and improved neurobehavioral score and sensory motor function in SHR rats. Conclusion: These findings suggest that augmented WNK1/4-NKCC1 signaling is responsible, at least in part, for the increased susceptibility to ischemic brain damage associated with hypertension. They highlight the potential for inhibition of WNK1/4-NKCC1 signaling as part of anti-ischemic stroke therapy. Supported by NIH Grant R01 NS38118 (D Sun).

WNK-Cab39-NKCC1 signaling increases the susceptibility to ischemic brain damage in hypertensive rats

With-no-lysine kinase (WNK) and Na+-K+-2Cl− cotransporter 1 (NKCC1) are involved in the pathogenesis of hypertension. In this study, we investigated the WNK-NKCC1 signaling pathway in spontaneously hypertensive rats (SHR) and their associated susceptibility to stroke injury. Basal NKCC1 protein levels were higher in SHR than in normotensive Wistar Kyoto (WKY) rat brains. After inducing ischemic stroke, adult male WKY and SHR received either saline or NKCC1 inhibitor bumetanide (10 mg/kg/day, i.p.) starting at 3-h post-reperfusion. NKCC1 inhibition blunted the extent of ischemic infarction in SHR and improved their neurobehavioral functions. Interestingly, ischemia led to increased NKCC1 phosphorylation in SHR but not in WKY rats. Pronounced elevation of WNK1, WNK2 and WNK4 protein and downregulation of WNK3 were detected in ischemic SHR, but not in ischemic WKY rats. Upregulation of WNK-NKCC1 complex in ischemic SHR brain was associated with increased Ca2+-binding protein 39 (Cab39), without increases in Ste20-related proline alanine-rich kinase or oxidative stress-responsive kinase-1. Moreover, subacute middle cerebral artery stroke human brain autopsy exhibited increased expression of NKCC1 protein. We conclude that augmented WNK-Cab39-NKCC1 signaling in SHR is associated with an increased susceptibility to ischemic brain damage and may serve as a novel target for anti-hypertensive and anti-ischemic stroke therapy.

Aldosterone modulates thiazide-sensitive sodium chloride cotransporter abundance via DUSP6-mediated ERK1/2 signaling pathway.

Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20-related proline alanine-rich kinase) or WNK4-extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone-mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2-specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs.

Mechanisms of angiotensin II stimulation of NCC are time-dependent in mDCT15 cells.

Angiotensin II (ANG II) increases thiazide-sensitive sodium-chloride cotransporter (NCC) activity both acutely and chronically. ANG II has been implicated as a switch that turns WNK4 from an inhibitor of NCC into an activator of NCC, and ANG II's effect on NCC appears to require WNK4. Chronically, ANG II stimulation of NCC results in an increase in total and phosphorylated NCC, but the role of NCC phosphorylation in acute ANG II actions is unclear. Here, using a mammalian cell model with robust native NCC activity, we corroborate the role that ANG II plays in WNK4 regulation and clarify the role of Ste20-related proline alanine-rich kinase (SPAK)-induced NCC phosphorylation in ANG II action. ANG II was noted to have a biphasic effect on NCC, with a peak increase in NCC activity in the physiologic range of 10(-11) M ANG II. This effect was apparent as early as 15 min and remained sustained through 120 min. These changes correlated with significant increases in NCC surface protein expression. Knockdown of WNK4 expression sharply attenuated the effect of ANG II. SPAK knockdown did not affect ANG II action at early time points (15 and 30 min), but it did attenuate the response at 60 min. Correspondingly, NCC phosphorylation did not increase at 15 or 30 min, but increased significantly at 60 min. We therefore conclude that within minutes of an increase in ANG II, NCC is rapidly trafficked to the cell surface in a phosphorylation-independent but WNK4-dependent manner. Then, after 60 min, ANG II induces SPAK-dependent phosphorylation of NCC.

Fungal communication requires the MAK-2 pathway elements STE-20 and RAS-2, the NRC-1 adapter STE-50 and the MAP kinase scaffold HAM-5.

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes.

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na(+) transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na(+)-K(+)-2Cl(-) cotransporter, NKCC2.

S8-5 Diabetic renal remodeling: Role of hydrogen sulfide

Matrix mettaloproteinase-9 (MMP-9) and H2S has been shown to modulate pathogenesis of diabetic nephropathy. However, the regulation of these molecules and their cross-talk in diabetic kidney is still unclear. Using WT, diabetic (Akita), MMP-9 (-/-), double knockout (DKO) of Akita/MMP-9(-/-) mice, and in vitro cell culture we have previously reported that MMP-9-mediated H2S generation triggered renovascular remodeling in diabetes. In hyperglycemic mice increased oxidative stress, induction of MMP-9, alteration in microRNAs expressions, and diminished expressions of CBS and CSE with concomitant decreased production of H2S were measured. In addition, increased expressions of NMDA-R1 and connexins (Cxs)-40 and -43 were also observed in diabetic kidney. In DKO mice, all the above molecules showed a trend toward baseline despite hyperglycemia. In vitro, glomerular endothelial cells treated with high glucose showed induction of MMP-9, attenuated H2S production, NMDA-R1 induction, and dysregulated Cxs expressions. Silencing MMP-9 by siRNA or inhibition of NMDA-R1 by MK801 or H2S treatment preserved Cxs-40 and -43. Our ongoing studies have revealed that deregulation of poly-(ADP-ribose)-polymerase 1 (PARP1) in hyperglycemic condition enhanced extracellular matrix (ECM) synthesis resulting in accumulation of matrix proteins. Interestingly, H2S treatment mitigated matrix remodeling. Furthermore, H2S-mediated formation of STRAD/MO25/LKB1 tri-molecular complex [STE20-related protein (STRAD), mouse protein-25, and liver kinase B1 (LKB1)] led the activation of AMP-K (adenosine monophosphate-activated protein kinase), and modulated autophagy in hyperglycemia. Together, these results suggest that in diabetic renovascular remodeling MMP-9 plays a major role and that H2S mitigates adverse remodeling.